Methods of producing 6-carbon chemicals via CoA-dependent carbon chain elongation associated with carbon storage

ABSTRACT

This document describes biochemical pathways for producing adipic acid, caprolactam, 6-aminohexanoic acid, 6-hydroxyhexanoic acid, hexamethylenediamine or 1,6-hexanediol by forming two terminal functional groups, comprised of carboxyl, amine or hydroxyl groups, in a C6 aliphatic backbone substrate. These pathways, metabolic engineering and cultivation strategies described herein rely on CoA-dependent elongation enzymes or analogs enzymes associated with the carbon storage pathways from polyhydroxyalkanoate accumulating bacteria.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.13/715,981, filed Dec. 14, 2012, which claims priority to U.S.Application Ser. No. 61/576,401, filed Dec. 16, 2011. The disclosures ofthe applications are incorporated by reference in their entirety.

TECHNICAL FIELD

This invention relates to methods for biosynthesizing one or more ofadipic acid, 6-aminohexanoic acid, 6-hydroxyhexanoic acid,hexamethylenediamine, caprolactam, and 1,6-hexanediol using one or moreisolated enzymes such as β-ketothiolases, dehydrogenases, reductases,hydratases, thioesterases, monooxygenases, and transaminases or usingrecombinant host cells expressing one or more such enzymes.

BACKGROUND

Nylons are polyamides that are generally synthesized by the condensationpolymerisation of a diamine with a dicarboxylic acid. Similarly, Nylonsmay be produced by the condensation polymerization of lactams. Aubiquitous nylon is Nylon 6,6, which is produced by reaction ofhexamethylenediamine (HMD) and adipic acid. Nylon 6 can be produced by aring opening polymerization of caprolactam. Therefore, adipic acid,hexamethylenediamine and caprolactam are important intermediates in theproduction of Nylons (Anton & Baird, Polyamides Fibers, Encyclopedia ofPolymer Science and Technology, 2001).

Industrially, adipic acid and caprolactam are produced via air oxidationof cyclohexane. The air oxidation of cyclohexane produces, in a seriesof steps, a mixture of cyclohexanone (K) and cyclohexanol (A),designated as KA oil. Nitric acid oxidation of KA oil produces adipicacid (Musser, Adipic acid, Ullmann's Encyclopedia of IndustrialChemistry, 2000). Caprolactam is produced from cyclohexanone via itsoxime and subsequent acid rearrangement (Fuchs, Kieczka and Moran,Caprolactam, Ullmann's Encyclopedia of Industrial Chemistry, 2000)

Industrially, hexamethylenediamine (HMD) is produced by hydrocyanationof C6 building block to adiponitrile, followed by hydrogenation to HMD(Herzog and Smiley, Hexamethylenediamine, Ullmann's Encyclopedia ofIndustrial Chemistry, 2012).

Given a reliance on petrochemical feedstocks; biotechnology offers analternative approach via biocatalysis. Biocatalysis is the use ofbiological catalysts, such as enzymes, to perform biochemicaltransformations of organic compounds.

Both bioderived feedstocks and petrochemical feedstocks are viablestarting materials for the biocatalysis processes.

Accordingly, against this background, it is clear that there is a needfor sustainable methods for producing one or more of adipic acid,caprolactam, 6-aminohexanoic acid, 6-hydroxyhexanoic acid,hexamethylenediamine, and 1,6-hexanediol (hereafter “C6 buildingblocks”) wherein the methods are biocatalyst based (Jang et al.,Biotechnology & Bioengineering, 2012, 109(10), 2437-2459).

However, no wild-type prokaryote or eukaryote naturally overproduces orexcretes C6 building blocks to the extracellular environment.Nevertheless, the metabolism of adipic acid and caprolactam has beenreported (Ramsay et al., Appl. Environ. Microbiol., 1986, 52(1),152-156; and Kulkarni and Kanekar, Current Microbiology, 1998, 37,191-194).

The dicarboxylic acid, adipic acid, is converted efficiently as a carbonsource by a number of bacteria and yeasts via β-oxidation into centralmetabolites. β-oxidation of Coenzyme A (CoA) activated adipate to CoAactivated 3-oxoadipate facilitates further catabolism via, for example,pathways associated with aromatic substrate degradation. The catabolismof 3-oxoadipyl-CoA to acetyl-CoA and succinyl-CoA by several bacteriaand fungi has been characterized comprehensively (Harwood and Parales,Annual Review of Microbiology, 1996, 50, 553-590). Both adipate and6-aminohexanoate are intermediates in the catabolism of caprolactam,finally degraded via 3-oxoadipyl-CoA to central metabolites.

Potential metabolic pathways have been suggested for producing adipicacid from biomass-sugar: (1) biochemically from glucose to cis,cismuconic acid via the ortho-cleavage aromatic degradation pathway,followed by chemical catalysis to adipic acid; (2) a reversible adipicacid degradation pathway via the condensation of succinyl-CoA andacetyl-CoA and (3) combining β-oxidation, a fatty acid synthase andω-oxidation. However, no information using these strategies has beenreported (Jang et al., Biotechnology & Bioengineering, 2012, 109(10),2437-2459).

The optimality principle states that microorganisms regulate theirbiochemical networks to support maximum biomass growth. Beyond the needfor expressing heterologous pathways in a host organism, directingcarbon flux towards C6 building blocks that serve as carbon sourcesrather than as biomass growth constituents, contradicts the optimalityprinciple. For example, transferring the 1-butanol pathway fromClostridium species into other production strains has often fallen shortby an order of magnitude compared to the production performance ofnative producers (Shen et al., Appl. Environ. Microbiol., 2011, 77(9),2905-2915).

The efficient synthesis of the six carbon aliphatic backbone precursoris a key consideration in synthesizing C6 building blocks prior toforming terminal functional groups, such as carboxyl, amine or hydroxylgroups, on the C6 aliphatic backbone.

SUMMARY

This document is based at least in part on the discovery that it ispossible to construct biochemical pathways for producing a six carbonchain aliphatic backbone precursor, in which two functional groups,e.g., carboxyl, amine or hydroxyl, can be formed, leading to thesynthesis of one or more of adipic acid, 6-aminohexanoic acid,6-hydroxyhexanoic acid, hexamethylenediamine, caprolactam, and1,6-hexanediol (hereafter “C6 building blocks”). Adipic acid andadipate, 6-hydroxyhexanoic acid and 6-hydroxyhexanoate, and6-aminohexanoic and 6-aminohexanoate are used interchangeably herein torefer to the compound in any of its neutral or ionized forms, includingany salt forms thereof. It is understood by those skilled in the artthat the specific form will depend on pH. These pathways, metabolicengineering, and cultivation strategies described herein rely onCoA-dependent elongation enzymes or homologs thereof associated with thecarbon storage pathways from polyhydroxyalkanoate accumulating bacteria.

In the face of the optimality principle, it surprisingly has beendiscovered that appropriate non-natural pathways, feedstocks, hostmicroorganisms, attenuation strategies to the host's biochemicalnetwork, and cultivation strategies may be combined to efficientlyproduce C6 building blocks.

In some embodiments, the C6 aliphatic backbone for conversion to a C6building block can be formed from acetyl-CoA via two cycles ofCoA-dependent carbon chain elongation using either NADH or NADPHdependent enzymes. See FIG. 1 and FIG. 2.

In some embodiments, the enzyme in the CoA-dependent carbon chainelongation pathway generating the C6 aliphatic backbone catalyzesirreversible enzymatic steps.

In some embodiments, the terminal carboxyl groups can be enzymaticallyformed using a thioesterase, an aldehyde dehydrogenase, a 6-oxohexanoatedehydrogenase, a 7-oxoheptanoate dehydrogenase, or a monooxygenase(e.g., in combination with an oxidoreductase and ferredoxin). See FIG. 3and FIG. 4. The thioesterase can have at least 70% sequence identity tothe amino acid sequence set forth in SEQ ID NO: 1.

In some embodiments, the terminal amine groups can be enzymaticallyformed using a ω-transaminase or a deacetylase. See FIG. 5 and FIG. 6.The ω-transaminase can have at least 70% sequence identity to any one ofthe amino acid sequences set forth in SEQ ID NOs. 7-12.

In some embodiments, the terminal hydroxyl group can be enzymaticallyformed using a monooxygenase (e.g., in combination with anoxidoreductase and ferredoxin), or an alcohol dehydrogenase. See FIG. 7and FIG. 8. The monooxygenase can have at least 70% sequence identity toany one of the amino acid sequences set forth in SEQ ID NO. 13-15.

In one aspect, this document features a method for biosynthesizing oneor more products selected from the group consisting of adipic acid,6-hydroxyhexanoic acid, 6-aminohexanoic acid, hexamethylenediamine,caprolactam, and 1,6-hexanediol. The method includes enzymaticallysynthesizing a six carbon chain aliphatic backbone (e.g., hexanoyl-CoA)and enzymatically forming, in one or more steps, two terminal functionalgroups selected from the group consisting of carboxyl, amine, andhydroxyl groups in the backbone to directly produce the product orproducing the product in a subsequent step. The two terminal functionalgroups can be the same (e.g., hydroxyl or amine) or can be different(e.g., a terminal hydroxyl group and a terminal carboxyl group; or aterminal amine and a terminal carboxyl group).

Hexanoyl-CoA can be enzymatically synthesized from acetyl-CoA via twocycles of CoA-dependent carbon chain elongation using either NADH orNADPH dependent enzymes. Hexanoyl-CoA can be formed by conversion ofhex-2-enoyl-CoA by an enoyl-CoA reductase classified under EC 1.3.1.44,EC 1.3.1.38, or EC 1.3.1.8 such as the gene product of ter or tdter.Hex-2-enoyl-CoA can be formed by conversion of (S) 3-hydroxyhexanoyl-CoAby a trans-2-enoyl-CoA hydratase classified under EC 4.2.1.17 or byconversion of (R) 3-hydroxyhexanoyl-CoA by a trans-2-enoyl-CoA hydrataseclassified under EC 4.2.1.119. The trans-2-enoyl-CoA hydratase can bethe gene product of crt. (S) 3-hydroxyhexanoyl-CoA can be formed byconversion of 3-oxohexanoyl-CoA by a 3-hydroxyacyl-CoA dehydrogenaseclassified under EC 1.1.1.35 such as the 3-hydroxyacyl-CoA dehydrogenaseencoded by fadB. The 3-oxohexanoyl-CoA can be formed by conversion ofbutanoyl-CoA by a β-ketothiolase classified under EC 2.3.1.16 such asthat encoded by bktB. Butanoyl-CoA can be formed by conversion ofcrotonyl-CoA by an enoyl-CoA reductase classified under EC 1.3.1.44, EC1.3.1.38, or EC 1.3.1.8. Crotonyl-CoA can be formed by conversion of (S)3-hydroxybutanoyl-CoA by a trans-2-enoyl-CoA hydratase classified underEC 4.2.1.17. The (S) 3-hydroxybutanoyl-CoA can be formed by conversionof acetoacetyl-CoA by a 3-hydroxybutyryl-CoA dehydrogenase classifiedunder EC 1.1.1.157 such as a 3-hydroxybutyryl-CoA dehydrogenase encodedby hbd. The acetoacetyl-CoA can be formed by conversion of acetyl-CoA bya β-ketothiolase classified under EC 2.3.1.9 such as that encoded byatoB or phaA. The acetoacetyl-CoA can be formed by conversion ofmalonyl-CoA by an acetoacetyl-CoA synthase classified under EC2.3.1.194. The malonyl-CoA can be formed by conversion of acetyl-CoA byan acetyl-CoA carboxylase classified under EC 6.4.1.2.

The (R) 3-hydroxyhexanoyl-CoA can be formed by conversion of3-oxohexanoyl-CoA by a 3-oxoacyl-CoA reductase classified under EC1.1.1.100 such as that encoded by fabG. The crotonyl-CoA can be formedby conversion of (R) 3-hydroxybutanoyl-CoA by a trans-2-enoyl-CoAhydratase classified under EC 4.2.1.119. The trans-2-s enoyl-CoAhydratase can be the gene product of phaJ. (R) 3-hydroxybutanoyl-CoA canbe formed by conversion of acetoacetyl-CoA by an acetoacyl-CoA reductaseclassified under EC 1.1.1.36 such as that encoded by phaB.

In any of the methods described herein, the method can include producinghexanoate by forming a first terminal carboxyl group in hexanoyl CoAusing a thioesterase and an aldehyde dehydrogenase, or a thioesterase.The thioesterase can be encoded by YciA, tesB or Acot13. Thethioesterase can have at least 70% sequence identity to the amino acidsequence set forth in SEQ ID NO: 1.

Hexanoate can be produced by forming a first terminal carboxyl group inhexanal by an aldehyde dehydrogenase classified under EC 1.2.1.4.Hexanal can be formed by conversion of hexanoyl-CoA by a butanaldehydrogenase classified under EC 1.2.1.57.

A carboxylate reductase (e.g., in combination with a phosphopantetheinyltransferase) can form a terminal aldehyde group as an intermediate informing the product. The carboxylate reductase can have at least 70%sequence identity to any one of the amino acid sequences set forth inSEQ ID NO. 2-6.

In any of the methods described herein, the methods can includeconverting hexanoate to adipic acid, 6-aminohexanoic acid,hexamethylenediamine, 6-hydroxyhexanoic acid, ε caprolactam or 1,6hexanediol with one or more enzymatic conversions, wherein one of theenzymatic conversions introduces the second terminal functional group.The method can include converting hexanoate to 6-hydroxyhexanoate usinga monooxygenase such as from the family CYP153 such as CYP153A.6-hydroxyhexanoate can be converted to adipate semialdehyde using (i) analcohol dehydrogenase such as encoded by YMR318C, a 5-hydroxypentanoatedehydrogenase such as encoded by cpnD or a 4-hydroxybutyratedehydrogenase such as encoded by gabD (ii) a 6-hydroxyhexanoatedehydrogenase such as that encoded by ChnD, or (iii) a monooxygenase inthe cytochrome P450 family.

In any of the methods described herein, adipic acid can be produced byforming the second terminal functional group in adipate semialdehydeusing (i) an aldehyde dehydrogenase classified under EC 1.2.1.3, (ii) a6-oxohexanoate dehydrogenase classified under EC 1.2.1.63 such as thatencoded by ChnE or a 7-oxoheptanoate dehydrogenase classified under EC1.2.1.- (e.g., the gene product of ThnG) or iii) a monooxygenase in thecytochrome P450 family.

In any of the methods described herein, 6-aminohexanoic acid can beproduced by forming the second terminal functional group in adipatesemialdehyde using a ω-transaminase classified under EC 2.61.18, EC2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82.

In any of the methods described herein, caprolactam can be produced from6-aminohexanoic acid using a lactamase classified under EC 3.5.2.-. Theamide bond associated with caprolactam is produced from a terminalcarboxyl group and terminal amine group of 6-aminohexanoate.

In any of the methods described herein, hexamethylenediamine can beproduced by forming a second terminal functional group in (i)6-aminohexanal using a ω-transaminase classified under EC 2.61.18, EC2.6.1.19, EC 2.6.1.29, EC 2.6.1.48 or EC 2.6.1.82 or in (ii)N6-acetyl-1,6-diaminohexane using a deacetylase classified, for example,under EC 3.5.1.17.

In any of the methods described herein, 1,6 hexanediol can be producedby forming the second terminal functional group in 6-hydroxyhexanalusing an alcohol dehydrogenase classified under EC 1.1.1.- (e.g., 1, 2,21, or 184) such as that encoded by YMR318C, YqhD or CAA81612.1.

In some embodiments, the biological feedstock can be or can derive from,monosaccharides, disaccharides, lignocellulose, hemicellulose,cellulose, lignin, levulinic acid and formic acid, triglycerides,glycerol, fatty acids, agricultural waste, condensed distillers'solubles, or municipal waste.

In some embodiments, the non-biological feedstock can be or can derivefrom natural gas, syngas, CO₂/H₂, methanol, ethanol, benzoate,non-volatile residue (NVR) or a caustic wash waste stream fromcyclohexane oxidation processes, or terephthalic acid/isophthalic acidmixture waste streams.

In some embodiments, the host microorganism is a prokaryote. Forexample, the prokaryote can be from the bacterial genus Escherichia suchas Escherichia coli; from the bacterial genus Clostridia such asClostridium ljungdahlii, Clostridium autoethanogenum or Clostridiumkluyveri; from the bacterial genus Corynebacteria such asCorynebacterium glutamicum; from the bacterial genus Cupriavidus such asCupriavidus necator or Cupriavidus metallidurans; from the bacterialgenus Pseudomonas such as Pseudomonas fluorescens, Pseudomonas putida orPseudomonas oleavorans; from the bacterial genus Delftia such as Delftiaacidovorans; from the bacterial genus Bacillus such as Bacillussubtillis; from the bacterial genus Lactobacillus such as Lactobacillusdelbrueckii; or from the bacterial genus Lactococcus such as Lactococcuslactis. Such prokaryotes also can be sources of genes for constructingrecombinant host cells described herein that are capable of producing C6building blocks.

In some embodiments, the host microorganism is a eukaryote (e.g., afungus such as a yeast). For example, the eukaryote can be from thefungal genus Aspergillus such as Aspergillus niger; from the yeast genusSaccharomyces such as Saccharomyces cerevisiae; from the yeast genusPichia such as Pichia pastoris; from the yeast genus Yarrowia such asYarrowia lipolytica; from the yeast genus Issatchenkia such asIssathenkia orientalis; from the yeast genus Debaryomyces such asDebaryomyces hansenii; from the yeast genus Arxula such as Arxulaadenoinivorans; or from the yeast genus Kluyveromyces such asKluyveromyces lactis. Such eukaryotes also can be sources of genes forconstructing recombinant host cells described herein that are capable ofproducing C6 building blocks.

In some embodiments, the host microorganism's tolerance to highconcentrations of one or more C6 building blocks is improved throughcontinuous cultivation in a selective environment.

In some embodiments, the host microorganism's biochemical network isattenuated or augmented to (1) ensure the intracellular availability ofacetyl-CoA, (2) create an NADH or NADPH imbalance that may only bebalanced via the formation of one or more C6 building blocks, (3)prevent degradation of central metabolites, central precursors leadingto and including C6 building blocks and (4) ensure efficient efflux fromthe cell.

In some embodiments, a non-cyclical cultivation strategy is used toachieve anaerobic, micro-aerobic, or aerobic cultivation conditions.

In some embodiments, a cyclical cultivation strategy is used toalternate between anaerobic and aerobic cultivation conditions.

In some embodiments, the cultivation strategy includes limitingnutrients, such as limiting nitrogen, phosphate or oxygen.

In some embodiments, one or more C6 building blocks are produced by asingle type of microorganism, e.g., a recombinant host containing one ormore exogenous nucleic acids, using a non-cyclical or cyclicalfermentation strategy.

In some embodiments, one or more C6 building blocks are produced byco-culturing more than one type of microorganism, e.g., two or moredifferent recombinant hosts, with each host containing a particular setof exogenous nucleic acids.

In some embodiments, one or more C6 building blocks can be produced bysuccessive fermentations, where the broth or centrate from the precedingfermentation can be fed to a succession of fermentations as a source offeedstock, central metabolite or central precursor; finally producingthe C6 building block.

This document also features a recombinant host comprising at least oneexogenous nucleic acid encoding, for example, one or more of a formatedehydrogenase, enoyl-CoA reductase, trans-2-enoyl-CoA hydratase,3-hydroxybutyryl-CoA dehydrogenase, β-ketothiolase, acetoacyl-CoAreductase, acetyl-CoA synthetase, acetyl-CoA carboxylase, a malicenzyme, puridine nucleotide transhydrogenase,glyceraldehyde-3P-dehydrogenase, thioesterase, aldehyde dehydrogenase,monooxygenase, alcohol dehydrogenase, 6-hydroxyhexanoate dehydrogenase,5-hydroxypentanoate dehydrogenase, 4-hydroxybutyrate dehydrogenase,6-oxohexanoate dehydrogenase, 7-oxoheptanoate dehydrogenase,ω-transaminase, propionyl-CoA synthetase, and a carboxylate reductase,wherein said host comprises one or more deficiencies, for example, inglucose-6-phosphate isomerase, acetate kinase, an enzyme degradingpyruvate to lactate such as lactate dehydrogenase, enzymes mediating thedegradation of phophoenolpyruvate to succinate such asmenaquinol-fumarate oxidoreductase, alcohol dehydrogenase, pyruvatedecarboxylase, 2-oxoacid decarboxylase, triose phosphate isomerase, or aNADH-specific glutamate dehydrogenase.

In one aspect, this document features a recombinant host that includesat least one exogenous nucleic acid encoding (i) a β-ketothiolase or anacetyl-CoA carboxylase and an acetoacetyl-CoA synthase, (ii) a3-hydroxyacyl-CoA dehydrogenase or a 3-oxoacyl-CoA reductase, (iii) anenoyl-CoA hydratase, and (iv) a trans-2-enoyl-CoA reductase, where thehost produces hexanoyl-CoA. The host further can include one or more ofa thioesterase, an aldehyde dehydrogenase, or a butanal dehydrogenase,wherein the host produces hexanal or hexanoate.

A recombinant producing hexanal or hexanoate further can include one ormore of a monooxygenase, an alcohol dehydrogenase, an aldehydedehydrogenase, a 6-hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoatedehydrogenase, a 4-hydroxybutyrate dehydrogenase, a 6-oxohexanoatedehydrogenase, or a 7-oxoheptanoate dehydrogenase, wherein the hostproduces adipic acid or adipate semialdehyde.

A recombinant producing hexanal or hexanoate further can include one ormore of monooxygenase, a transaminase, a 6-hydroxyhexanoatedehydrogenase, a 5-hydroxypentanoate dehydrogenase, a 4-hydroxybutyratedehydrogenase, and an alcohol dehydrogenase, wherein the host produces6-aminohexanoate. The host further can include a lactamase and producecaprolactam.

A recombinant producing hexanal or hexanoate further can include amonooxygenase, the host producing 6-hydroxyhexanoic acid.

A recombinant host producing hexanal, hexanoate, 6-hydroxyhexanoate, or6-aminohexanoate further can include one or more of a carboxylatereductase, a ω-transaminase, a deacetylase, a N-acetyl transferase, oran alcohol dehydrogenase, and produce hexamethylenediamine.

A recombinant host producing 6-hydroxyhexanoate further can include acarboxylate reductase or an alcohol dehydrogenase, the host producing1,6-hexanediol.

Any of the recombinant hosts described herein further can include one ormore of the following attenuated enzymes: a polyhydroxyalkanoatesynthase, an acetyl-CoA thioesterase, a phosphotransacetylase formingacetate, an acetate kinase, a lactate dehydrogenase, amenaquinol-fumarate oxidoreductase, a 2-oxoacid decarboxylase producingisobutanol, an alcohol dehydrogenase forming ethanol, a triose phosphateisomerase, a pyruvate decarboxylase, a glucose-6-phosphate isomerase,NADH-consuming transhydrogenase, an NADH-specific glutamatedehydrogenase, a NADH/NADPH-utilizing glutamate dehydrogenase, apimeloyl-CoA dehydrogenase; an acyl-CoA dehydrogenase accepting C6building blocks and central precursors as substrates; a butaryl-CoAdehydrogenase; or an adipyl-CoA synthetase.

Any of the recombinant hosts described herein further can overexpressone or more genes encoding: an acetyl-CoA synthetase, a6-phosphogluconate dehydrogenase; a transketolase; a puridine nucleotidetranshydrogenase; a glyceraldehyde-3P-dehydrogenase; a malic enzyme; aglucose-6-phosphate dehydrogenase; a glucose dehydrogenase; a fructose1,6 diphosphatase; a L-alanine dehydrogenase; a L-glutamatedehydrogenase; a formate dehydrogenase; a L-glutamine synthetase; adiamine transporter; a dicarboxylate transporter; and/or a multidrugtransporter.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims. The word “comprising” inthe claims may be replaced by “consisting essentially of” or with“consisting of,” according to standard practice in patent law.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic of an exemplary biochemical pathway leading tohexanoyl-CoA using NADH-dependent enzymes and with acetyl-CoA as acentral metabolite.

FIG. 2 is a schematic of an exemplary biochemical pathway leading tohexanoyl-CoA using NADPH-dependent enzymes and with acetyl-CoA as acentral metabolite.

FIG. 3 is a schematic of exemplary biochemical pathways leading tohexanoate using hexanoyl-CoA as a central precursor.

FIG. 4 is a schematic of exemplary biochemical pathways leading toadipic acid using hexanoate as a central precursor.

FIG. 5 is a schematic of an exemplary biochemical pathway leading to6-aminhexanoate using hexanoate as a central precursor and a schematicof an exemplary biochemical pathway leading to caprolactam from6-aminohexanoate.

FIG. 6 is a schematic of exemplary biochemical pathways leading tohexamethylenediamine using 6-aminohexanoate, 6-hydroxyhexanoate, oradipate semialdehyde as a central precursor.

FIG. 7 is a schematic of an exemplary biochemical pathway leading to6-hydroxyhexanoate using hexanoate as a central precursor.

FIG. 8 is a schematic of an exemplary biochemical pathway leading to1,6-hexanediol using 6-hydroxyhexanoate as a central precursor.

FIG. 9 contains the amino acid sequences of an Escherichia colithioesterase encoded by tesB (see GenBank Accession No. AAA24665.1, SEQID NO: 1), a Mycobacterium marinum carboxylate reductase (see GenbankAccession No. ACC40567.1, SEQ ID NO: 2), a Mycobacterium smegmatiscarboxylate reductase (see Genbank Accession No. ABK71854.1, SEQ ID NO:3), a Segniliparus rugosus carboxylate reductase (see Genbank AccessionNo. EFV11917.1, SEQ ID NO: 4), a Mycobacterium massiliense carboxylatereductase (see Genbank Accession No. EIV11143.1, SEQ ID NO: 5), aSegniliparus rotundus carboxylate reductase (see Genbank Accession No.ADG98140.1, SEQ ID NO: 6), a Chromobacterium violaceum ω-transaminase(see Genbank Accession No. AAQ59697.1, SEQ ID NO: 7), a Pseudomonasaeruginosa ω-transaminase (see Genbank Accession No. AAG08191.1, SEQ IDNO: 8), a Pseudomonas syringae ω-transaminase (see Genbank Accession No.AAY39893.1, SEQ ID NO: 9), a Rhodobacter sphaeroides ω-transaminase (seeGenbank Accession No. ABA81135.1, SEQ ID NO: 10), an Escherichia coliω-transaminase (see Genbank Accession No. AAA57874.1, SEQ ID NO: 11), aVibrio Fluvialis ω-transaminase (See Genbank Accession No. AEA39183.1,SEQ ID NO: 12); a Polaromonas sp. JS666 monooxygenase (see GenbankAccession No. ABE47160.1, SEQ ID NO:13), a Mycobacterium sp. HXN-1500monooxygenase (see Genbank Accession No. CAH04396.1, SEQ ID NO:14), aMycobacterium austroafricanum monooxygenase (see Genbank Accession No.ACJ06772.1, SEQ ID NO:15), a Polaromonas sp. JS666 oxidoreductase (seeGenbank Accession No. ABE47159.1, SEQ ID NO:16), a Mycobacterium sp.HXN-1500 oxidoreductase (see Genbank Accession No. CAH04397.1, SEQ IDNO:17), a Polaromonas sp. JS666 ferredoxin (see Genbank Accession No.ABE47158.1, SEQ ID NO:18), a Mycobacterium sp. HXN-1500 ferredoxin (seeGenbank Accession No. CAH04398.1, SEQ ID NO:19), a Bacillus subtilisphosphopantetheinyl transferase (see Genbank Accession No. CAA44858.1,SEQ ID NO:20), and a Nocardia sp. NRRL 5646 phosphopantetheinyltransferase (see Genbank Accession No. ABI83656.1, SEQ ID NO:21).

FIG. 10 is a bar graph of the relative absorbance at 412 nm of releasedCoA as a measure of the activity of a thioesterase for convertinghexanoyl-CoA to hexanoate relative to the empty vector control.

FIG. 11 is a bar graph summarizing the change in absorbance at 340 nmafter 20 minutes, which is a measure of the consumption of NADPH and theactivity of the carboxylate reductases of the enzyme only controls (nosubstrate).

FIG. 12 is a bar graph of the change in absorbance at 340 nm after 20minutes, which is a measure of the consumption of NADPH and the activityof carboxylate reductases for converting adipate to adipate semialdehyderelative to the empty vector control.

FIG. 13 is a bar graph of the change in absorbance at 340 nm after 20minutes, which is a measure of the consumption of NADPH and the activityof carboxylate reductases for converting 6-hydroxyhexanoate to6-hydroxyhexanal relative to the empty vector control.

FIG. 14 is a bar graph of the change in absorbance at 340 nm after 20minutes, which is a measure of the consumption of NADPH and the activityof carboxylate reductases for converting N6-acetyl-6-aminohexanoate toN6-acetyl-6-aminohexanal relative to the empty vector control.

FIG. 15 is a bar graph of the change in absorbance at 340 nm after 20minutes, which is a measure of the consumption of NADPH and activity ofcarboxylate reductases for converting adipate semialdehyde to hexanedialrelative to the empty vector control.

FIG. 16 is a bar graph summarizing the percent conversion after 4 hoursof pyruvate to L-alanine (mol/mol) as a measure of the ω-transaminaseactivity of the enzyme only controls (no substrate).

FIG. 17 is a bar graph of the percent conversion after 24 hours ofpyruvate to L-alanine (mol/mol) as a measure of the ω-transaminaseactivity for converting 6-aminohexanoate to adipate semialdehyderelative to the empty vector control.

FIG. 18 is a bar graph of the percent conversion after 4 hours ofL-alanine to pyruvate (mol/mol) as a measure of the ω-transaminaseactivity for converting adipate semialdehyde to 6-aminohexanoaterelative to the empty vector control.

FIG. 19 is a bar graph of the percent conversion after 4 hours ofpyruvate to L-alanine (mol/mol) as a measure of the ω-transaminaseactivity for converting hexamethylenediamine to 6-aminohexanal relativeto the empty vector control.

FIG. 20 is a bar graph of the percent conversion after 4 hours ofpyruvate to L-alanine (mol/mol) as a measure of the ω-transaminaseactivity for converting N6-acetyl-1,6-diaminohexane toN6-acetyl-6-aminohexanal relative to the empty vector control.

FIG. 21 is a bar graph of the percent conversion after 4 hours ofpyruvate to L-alanine (mol/mol) as a measure of the ω-transaminaseactivity for converting 6-aminohexanol to 6-oxohexanol relative to theempty vector control.

FIG. 22 is a bar graph of the change in peak area for 6-hydroxyhexanoateas determined via LC-MS, as a measure of the monooxygenase activity forconverting hexanoate to 6-hydroxyhexanoate relative to the empty vectorcontrol.

DETAILED DESCRIPTION

In general, this document provides enzymes, non-natural pathways,cultivation strategies, feedstocks, host microorganisms and attenuationsto the host's biochemical network, which generates a six carbon chainaliphatic backbone from central metabolites into which two terminalfunctional groups may be formed leading to the synthesis of one or moreof adipic acid, caprolactam, 6-aminohexanoic acid, 6-hydroxyhexanoicacid, hexamethylenediamine or 1,6-hexanediol (referred to as “C6building blocks” herein). As used herein, the term “central precursor”is used to denote any metabolite in any metabolic pathway shown hereinleading to the synthesis of a C6 building block. The term “centralmetabolite” is used herein to denote a metabolite that is produced inall microorganisms to support growth.

Host microorganisms described herein can include endogenous pathwaysthat can be manipulated such that one or more C6 building blocks can beproduced. In an endogenous pathway, the host microorganism naturallyexpresses all of the enzymes catalyzing the reactions within thepathway. A host microorganism containing an engineered pathway does notnaturally express all of the enzymes catalyzing the reactions within thepathway but has been engineered such that all of the enzymes within thepathway are expressed in the host.

The term “exogenous” as used herein with reference to a nucleic acid (ora protein) and a host refers to a nucleic acid that does not occur in(and cannot be obtained from) a cell of that particular type as it isfound in nature or a protein encoded by such a nucleic acid. Thus, anon-naturally-occurring nucleic acid is considered to be exogenous to ahost once in the host. It is important to note thatnon-naturally-occurring nucleic acids can contain nucleic acidsubsequences or fragments of nucleic acid sequences that are found innature provided the nucleic acid as a whole does not exist in nature.For example, a nucleic acid molecule containing a genomic DNA sequencewithin an expression vector is non-naturally-occurring nucleic acid, andthus is exogenous to a host cell once introduced into the host, sincethat nucleic acid molecule as a whole (genomic DNA plus vector DNA) doesnot exist in nature. Thus, any vector, autonomously replicating plasmid,or virus (e.g., retrovirus, adenovirus, or herpes virus) that as a wholedoes not exist in nature is considered to be non-naturally-occurringnucleic acid. It follows that genomic DNA fragments produced by PCR orrestriction endonuclease treatment as well as cDNAs are considered to benon-naturally-occurring nucleic acid since they exist as separatemolecules not found in nature. It also follows that any nucleic acidcontaining a promoter sequence and polypeptide-encoding sequence (e.g.,cDNA or genomic DNA) in an arrangement not found in nature isnon-naturally-occurring nucleic acid. A nucleic acid that isnaturally-occurring can be exogenous to a particular host microorganism.For example, an entire chromosome isolated from a cell of yeast x is anexogenous nucleic acid with respect to a cell of yeast y once thatchromosome is introduced into a cell of yeast y.

In contrast, the term “endogenous” as used herein with reference to anucleic acid (e.g., a gene) (or a protein) and a host refers to anucleic acid (or protein) that does occur in (and can be obtained from)that particular host as it is found in nature. Moreover, a cell“endogenously expressing” a nucleic acid (or protein) expresses thatnucleic acid (or protein) as does a host of the same particular type asit is found in nature. Moreover, a host “endogenously producing” or that“endogenously produces” a nucleic acid, protein, or other compoundproduces that nucleic acid, protein, or compound as does a host of thesame particular type as it is found in nature.

For example, depending on the host and the compounds produced by thehost, one or more of the following enzymes may be expressed in the hostin addition to (i) a β-ketothioase or an acetyl-CoA carboxylase andacetoacetyl-CoA synthase, (ii) a 3-hydroxyacyl-CoA dehydrogenase or a3-oxoacyl-CoA reductase, (iii) an enoyl-CoA hydratase, and (iv) atrans-2-enoyl-CoA reductase: a thioesterase, an aldehyde dehydrogenase,a butanal dehydrogenase, a monooxygenase, an alcohol dehydrogenase, a6-oxohexanoate dehydrogenase, a 7-oxoheptanoate dehydrogenase, a cotransaminase, a 6-hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoatedehydrogenase, a 4-hydroxybutyrate dehydrogenase, a carboxylatereductase, a deacetylase, or an N-acetyl transferase. In recombinanthosts expressing a carboxylate reductase, a phosphopantetheinyltransferase also can be expressed as it enhances activity of thecarboxylate reductase. In recombinant hosts expressing a monooxygenase,an electron transfer chain protein such as an oxidoreductase orferredoxin polypeptide also can be expressed.

In some embodiments, a recombinant host can include at least oneexogenous nucleic acid encoding a β-ketothioase or an acetyl-CoAcarboxylase and acetoacetyl-CoA synthase, a 3-hydroxyacyl-CoAdehydrogenase or a 3-oxoacyl-CoA reductase, an enoyl-CoA hydratase, anda trans-2-enoyl-CoA reductase, and produce hexanoyl-CoA. Such a hostfurther can include one or more of (e.g., two or three of) athioesterase, an aldehyde dehydrogenase, or a butanal dehydrogenase, andproduce hexanal or hexanoate.

A recombinant host producing hexanal or hexanoate further can includeone or more of a monooxygenase, an alcohol dehydrogenase, an aldehydedehydrogenase, a 6-hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoatedehydrogenase, a 4-hydroxybutyrate dehydrogenase, a 6-oxohexanoatedehydrogenase, or a 7-oxoheptanoate dehydrogenase, and produce adipicacid or adipate semialdehyde. For example, a recombinant host furthercan include a monooxygenase and produce adipic acid or adipatesemialdehyde. As another example, a recombinant host further can include(i) a monooxygenase, (ii) an alcohol dehydrogenase, a 6-hydroxyhexanoatedehydrogenase, a 5-hydroxypentanoate dehydrogenase or a4-hydroxybutyrate dehydrogenase and (iii) an aldehyde dehydrogenase, a6-oxohexanoate dehydrogenase, or a 7-oxoheptanoate dehydrogenase, andproduce adipic acid.

A recombinant host producing hexanal or hexanoate further can includeone or more of a monooxygenase, a transaminase, a 6-hydroxyhexanoatedehydrogenase, a 5-hydroxypentanoate dehydrogenase, a 4-hydroxybutyratedehydrogenase, and an alcohol dehydrogenase, and produce6-aminohexanoate. For example, a recombinant host further can includeeach of a monooxygenase, a transaminase, and a 6-hydroxyhexanoatedehydrogenase.

A recombinant host producing hexanal or hexanoate further can include amonooxygenase, and produce 6-hydroxyheptanoic acid.

A recombinant host producing 6-aminohexanoate, 6-hydroxyhexanoate, oradipate semialdehyde further can include one or more of a carboxylatereductase, a ω-transaminase, a deacetylase, a N-acetyl transferase, oran alcohol dehydrogenase, and produce hexamethylenediamine. In someembodiments, a recombinant host further can include each of acarboxylate reductase, a ω-transaminase, a deacetylase, and an N-acetyltransferase. In some embodiments, a recombinant host further can includea carboxylate reductase and a ω-transaminase. In some embodiments, arecombinant host further can include a carboxylate reductase, aω-transaminase, and an alcohol dehydrogenase.

A recombinant host producing 6-hydroxyhexanoic acid further can includeone or more of a carboxylate reductase and an alcohol dehydrogenase, andproduce 1,6-hexanediol.

Within an engineered pathway, the enzymes can be from a single source,i.e., from one species or genera, or can be from multiple sources, i.e.,different species or genera. Nucleic acids encoding the enzymesdescribed herein have been identified from various organisms and arereadily available in publicly available databases such as GenBank orEMBL.

Any of the enzymes described herein that can be used for production ofone or more C6 building blocks can have at least 70% sequence identity(homology) (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or100%) to the amino acid sequence of the corresponding wild-type enzyme.For example, a thioesterase described herein can have at least 70%sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 95%,97%, 98%, 99%, or 100%) to the amino acid sequence of an Escherichiacoli thioesterase encoded by tesB (see GenBank Accession No. AAA24665.1,SEQ ID NO: 1). See FIG. 9.

For example, a carboxylate reductase described herein can have at least70% sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%,95%, 97%, 98%, 99%, or 100%) to the amino acid sequence of aMycobacterium marinum (see Genbank Accession No. ACC40567.1, SEQ ID NO:2), a Mycobacterium smegmatis (see Genbank Accession No. ABK71854.1, SEQID NO: 3), a Segniliparus rugosus (see Genbank Accession No. EFV11917.1,SEQ ID NO: 4), a Mycobacterium massiliense (see Genbank Accession No.EIV 11143.1, SEQ ID NO: 5), or a Segniliparus rotundus (see GenbankAccession No. ADG98140.1, SEQ ID NO: 6) carboxylate reductase. See, FIG.9.

For example, a ω-transaminase described herein can have at least 70%sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 95%,97%, 98%, 99%, or 100%) to the amino acid sequence of a Chromobacteriumviolaceum (see Genbank Accession No. AAQ59697.1, SEQ ID NO: 7), aPseudomonas aeruginosa (see Genbank Accession No. AAG08191.1, SEQ ID NO:8), a Pseudomonas syringae (see Genbank Accession No. AAY39893.1, SEQ IDNO: 9), a Rhodobacter sphaeroides (see Genbank Accession No. ABA81135.1,SEQ ID NO: 10), an Escherichia coli (see Genbank Accession No.AAA57874.1, SEQ ID NO: 11), or a Vibrio fluvialis (see Genbank AccessionNo. AEA39183.1, SEQ ID NO: 12) ω-transaminase. Some of thesew-transaminases are diamine ω-transaminases. See, FIG. 9.

For example, a monooxygenase described herein can have at least 70%sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 95%,97%, 98%, 99%, or 100%) to the amino acid sequence of a Polaromonas sp.JS666 monooxygenase (see Genbank Accession No. ABE47160.1, SEQ IDNO:13), a Mycobacterium sp. HXN-1500 monooxygenase (see GenbankAccession No. CAH04396.1, SEQ ID NO:14), or a Mycobacteriumaustroafricanum monooxygenase (see Genbank Accession No. ACJ06772.1, SEQID NO:15). See, FIG. 9.

For example, an oxidoreductase described herein can have at least 70%sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 95%,97%, 98%, 99%, or 100%) to the amino acid sequence of a Polaromonas sp.JS666 oxidoreductase (see Genbank Accession No. ABE47159.1, SEQ IDNO:16) or a Mycobacterium sp. HXN-1500 oxidoreductase (see GenbankAccession No. CAH04397.1, SEQ ID NO:17). See, FIG. 9.

For example, a ferredoxin polypeptide described herein can have at least70% sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%,95%, 97%, 98%, 99%, or 100%) to the amino acid sequence of a Polaromonassp. JS666 ferredoxin (see Genbank Accession No. ABE47158.1, SEQ IDNO:18) or a Mycobacterium sp. HXN-1500 ferredoxin (see Genbank AccessionNo. CAH04398.1, SEQ ID NO:19). See, FIG. 9.

For example, a phosphopantetheinyl transferase described herein can haveat least 70% sequence identity (homology) (e.g., at least 75%, 80%, 85%,90%, 95%, 97%, 98%, 99%, or 100%) to the amino acid sequence of aBacillus subtilis phosphopantetheinyl transferase (see Genbank AccessionNo. CAA44858.1, SEQ ID NO: 20) or a Nocardia sp. NRRL 5646phosphopantetheinyl transferase (see Genbank Accession No. ABI83656.1,SEQ ID NO:21). See, FIG. 9.

The percent identity (homology) between two amino acid sequences can bedetermined as follows. First, the amino acid sequences are aligned usingthe BLAST 2 Sequences (Bl2seq) program from the stand-alone version ofBLASTZ containing BLASTP version 2.0.14. This stand-alone version ofBLASTZ can be obtained from Fish & Richardson's web site (e.g.,www.fr.com/blast/) or the U.S. government's National Center forBiotechnology Information web site (www.ncbi.nlm.nih.gov). Instructionsexplaining how to use the Bl2seq program can be found in the readme fileaccompanying BLASTZ. Bl2seq performs a comparison between two amino acidsequences using the BLASTP algorithm. To compare two amino acidsequences, the options of Bl2seq are set as follows: -i is set to a filecontaining the first amino acid sequence to be compared (e.g.,C:\seq1.txt); -j is set to a file containing the second amino acidsequence to be compared (e.g., C:\seq2.txt); -p is set to blastp; -o isset to any desired file name (e.g., C:\output.txt); and all otheroptions are left at their default setting. For example, the followingcommand can be used to generate an output file containing a comparisonbetween two amino acid sequences: C:\Bl2seq -i c:\seq1.txt -jc:\seq2.txt -p blastp -o c:\output.txt. If the two compared sequencesshare homology (identity), then the designated output file will presentthose regions of homology as aligned sequences. If the two comparedsequences do not share homology (identity), then the designated outputfile will not present aligned sequences. Similar procedures can befollowing for nucleic acid sequences except that blastn is used.

Once aligned, the number of matches is determined by counting the numberof positions where an identical amino acid residue is presented in bothsequences. The percent identity (homology) is determined by dividing thenumber of matches by the length of the full-length polypeptide aminoacid sequence followed by multiplying the resulting value by 100. It isnoted that the percent identity (homology) value is rounded to thenearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 is roundeddown to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 is rounded upto 78.2. It also is noted that the length value will always be aninteger.

It will be appreciated that a number of nucleic acids can encode apolypeptide having a particular amino acid sequence. The degeneracy ofthe genetic code is well known to the art; i.e., for many amino acids,there is more than one nucleotide triplet that serves as the codon forthe amino acid. For example, codons in the coding sequence for a givenenzyme can be modified such that optimal expression in a particularspecies (e.g., bacteria or fungus) is obtained, using appropriate codonbias tables for that species.

Functional fragments of any of the enzymes described herein can also beused in the methods of the document. The term “functional fragment” asused herein refers to a peptide fragment of a protein that has at least25% (e.g., at least: 30%; 40%; 50%; 60%; 70%; 75%; 80%; 85%; 90%; 95%;98%; 99%; 100%; or even greater than 100%) of the activity of thecorresponding mature, full-length, wild-type protein. The functionalfragment can generally, but not always, be comprised of a continuousregion of the protein, wherein the region has functional activity.

This document also provides (i) functional variants of the enzymes usedin the methods of the document and (ii) functional variants of thefunctional fragments described above. Functional variants of the enzymesand functional fragments can contain additions, deletions, orsubstitutions relative to the corresponding wild-type sequences. Enzymeswith substitutions will generally have not more than 50 (e.g., not morethan one, two, three, four, five, six, seven, eight, nine, ten, 12, 15,20, 25, 30, 35, 40, or 50) amino acid substitutions (e.g., conservativesubstitutions). This applies to any of the enzymes described herein andfunctional fragments. A conservative substitution is a substitution ofone amino acid for another with similar characteristics. Conservativesubstitutions include substitutions within the following groups: valine,alanine and glycine; leucine, valine, and isoleucine; aspartic acid andglutamic acid; asparagine and glutamine; serine, cysteine, andthreonine; lysine and arginine; and phenylalanine and tyrosine. Thenonpolar hydrophobic amino acids include alanine, leucine, isoleucine,valine, proline, phenylalanine, tryptophan and methionine. The polarneutral amino acids include glycine, serine, threonine, cysteine,tyrosine, asparagine and glutamine. The positively charged (basic) aminoacids include arginine, lysine and histidine. The negatively charged(acidic) amino acids include aspartic acid and glutamic acid. Anysubstitution of one member of the above-mentioned polar, basic or acidicgroups by another member of the same group can be deemed a conservativesubstitution. By contrast, a nonconservative substitution is asubstitution of one amino acid for another with dissimilarcharacteristics.

Deletion variants can lack one, two, three, four, five, six, seven,eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acidsegments (of two or more amino acids) or non-contiguous single aminoacids. Additions (addition variants) include fusion proteins containing:(a) any of the enzymes described herein or a fragment thereof; and (b)internal or terminal (C or N) irrelevant or heterologous amino acidsequences. In the context of such fusion proteins, the term“heterologous amino acid sequences” refers to an amino acid sequenceother than (a). A heterologous sequence can be, for example a sequenceused for purification of the recombinant protein (e.g., FLAG,polyhistidine (e.g., hexahistidine), hemagglutinin (HA),glutathione-S-transferase (GST), or maltosebinding protein (MBP)).Heterologous sequences also can be proteins useful as detectablemarkers, for example, luciferase, green fluorescent protein (GFP), orchloramphenicol acetyl transferase (CAT). In some embodiments, thefusion protein contains a signal sequence from another protein. Incertain host cells (e.g., yeast host cells), expression and/or secretionof the target protein can be increased through use of a heterologoussignal sequence. In some embodiments, the fusion protein can contain acarrier (e.g., KLH) useful, e.g., in eliciting an immune response forantibody generation) or ER or Golgi apparatus retention signals.Heterologous sequences can be of varying length and in some cases can bea longer sequences than the full-length target proteins to which theheterologous sequences are attached.

Engineered hosts can naturally express none or some (e.g., one or more,two or more, three or more, four or more, five or more, or six or more)of the enzymes of the pathways described herein. Thus, a pathway withinan engineered host can include all exogenous enzymes, or can includeboth endogenous and exogenous enzymes. Endogenous genes of theengineered hosts also can be disrupted to prevent the formation ofundesirable metabolites or prevent the loss of intermediates in thepathway through other enzymes acting on such intermediates. Engineeredhosts can be referred to as recombinant hosts or recombinant host cells.As described herein recombinant hosts can include nucleic acids encodingone or more of a dehydrogenase, a β-ketothiolase, a acetoacetyl-CoAsynthase, a carboxylase, a reductase, a hydratase, a thioesterase, amonooxygenase, a thioesterase, or transaminase as described herein.

In addition, the production of C6 building blocks can be performed invitro using the isolated enzymes described herein, using a lysate (e.g.,a cell lysate) from a host microorganism as a source of the enzymes, orusing a plurality of lysates from different host microorganisms as thesource of the enzymes.

The reactions of the pathways described herein can be performed in oneor more host strains (a) naturally expressing one or more relevantenzymes, (b) genetically engineered to express one or more relevantenzymes, or (c) naturally expressing one or more relevant enzymes andgenetically engineered to express one or more relevant enzymes.Alternatively, relevant enzymes can be extracted from of the above typesof host cells and used in a purified or semi-purified form. Moreover,such extracts include lysates (e.g. cell lysates) that can be used assources of relevant enzymes. In the methods provided by the document,all the steps can be performed in host cells, all the steps can beperformed using extracted enzymes, or some of the steps can be performedin cells and others can be performed using extracted enzymes.

Enzymes Generating the C6 Aliphatic Backbone for Conversion to C6Building Blocks

As depicted in FIG. 1 and FIG. 2, the C6 aliphatic backbone forconversion to C6 building blocks can be formed from acetyl-CoA via twocycles of CoA-dependent carbon chain elongation using either NADH orNADPH dependent enzymes.

In some embodiments, a CoA-dependent carbon chain elongation cyclecomprises a β-ketothiolase or an acetyl-CoA carboxylase and anacetoacetyl-CoA synthase, a 3-hydroxyacyl-CoA dehydrogenase or3-oxoacyl-CoA reductase, an enoyl-CoA hydratase and a trans-2-enoyl-CoAreductase. A β-ketothiolase can convert acetyl-CoA to acetoacetyl-CoAand can convert butanoyl-CoA to 3-oxohexanoyl-CoA. Acetyl-CoAcarboxylase can convert acetyl-CoA to malonyl-CoA. An acetoacetyl-CoAsynthase can convert malonyl-CoA to acetoacetyl-CoA. A3-hydroxybutyryl-CoA dehydrogenase can convert acetoacetyl-CoA to3-hydroxybutanoyl CoA. A 3-oxoacyl-CoA reductase/3-hydroxyacyl-CoAdehydrogenase can convert 3-oxohexanoyl-CoA to 3-hydroxyhexanoyl-CoA. Anenoyl-CoA hydratase can convert 3-hydroxybutanoyl-CoA to crotonyl-CoAand can convert 3-hydroxyhexanoyl-CoA to hex-2-enoyl-CoA. Atrans-2-enoyl-CoA reductase can convert crotonyl-CoA to butanoyl-CoA andcan convert hex-2-enoyl-CoA to hexanoyl-CoA.

In some embodiments, a β-ketothiolase may be classified under EC2.3.1.9, such as the gene product of atoB or phaA, or classified underEC 2.3.1.16, such as the gene product of bktB. The β-ketothiolaseencoded by bktB from Cupriavidus necator accepts acetyl-CoA andbutanoyl-CoA as substrates, forming the CoA-activated C6 aliphaticbackbone (see, e.g., Haywood et al., FEMS Microbiology Letters, 1988,52:91-96; Slater et al., J. Bacteriol., 1998, 180(8):1979-1987). Theβ-ketothiolase encoded by atoB or phaA accepts acetyl-CoA as substrates,forming butanoyl-CoA (see, Haywood et al., 1988, supra; Slater et al.,1998, supra).

In some embodiments, an acetyl-CoA carboxylase can be classified, forexample, under EC 6.4.1.2. Conversion of acetyl-CoA to malonyl-CoA by anacetyl-CoA carboxylase has been shown to increase the rate of fatty acidsynthesis (Davis et al., J. Biol. Chem., 2000, 275(37), 28593-28598).

In some embodiments, an acetoacetyl-CoA synthase may be classified underEC 2.3.1.194. It has been demonstrated that acetoacetyl-CoA synthase maybe used as an irreversible substitute for the gene product of phaA inthe carbon chain elongation associated with polyhydroxybutyratesynthesis (Matsumoto et al., Biosci. Biotechnol. Biochem., 2011, 75(2),364-366).

In some embodiments, a 3-hydroxyacyl-CoA dehydrogenase or 3-oxoacyl-CoAdehydrogenase can be classified under EC 1.1.1.-. For example, the3-hydroxyacyl-CoA dehydrogenase can be classified under EC 1.1.1.35,such as the gene product of fadB; classified under EC 1.1.1.157, such asthe gene product of hbd (also can be referred to as a3-hydroxybutyryl-CoA dehydrogenase); or classified under EC 1.1.1.36,such as the acetoacetyl-CoA reductase gene product of phaB (Liu & Chen,Appl. Microbiol. Biotechnol., 2007, 76(5):1153-1159; Shen et al., Appl.Environ. Microbiol., 2011, 77(9):2905-2915; Budde et al., J. Bacteriol.,2010, 192(20):5319-5328).

In some embodiments, a 3-oxoacyl-CoA reductase can be classified underEC 1.1.1.100, such as the gene product of fabG (Budde et al., J.Bacteriol., 2010, 192(20):5319-5328; Nomura et al., Appl. Environ.Microbiol., 2005, 71(8):4297-4306).

In some embodiments, an enoyl-CoA hydratase can be classified under EC4.2.1.17, such as the gene product of crt, or classified under EC4.2.1.119, such as the gene product of phaJ (Shen et al., 2011, supra;Fukui et al., J. Bacteriol., 1998, 180(3):667-673).

In some embodiments, a trans-2-enoyl-CoA reductase can be classifiedunder EC 1.3.1.38, EC 1.3.1.8, or EC 1.3.1.44, such as the gene productof ter (Nishimaki et al., J. Biochem., 1984, 95:1315-1321; Shen et al.,2011, supra) or tdter (Bond-Watts et al., Biochemistry, 2012,51:6827-6837).

Enzymes Generating the Terminal Carboxyl Groups in the Biosynthesis ofC6 Building Blocks

As depicted in FIG. 3 and FIG. 4, the terminal carboxyl groups can beenzymatically formed using a thioesterase, an aldehyde dehydrogenase, a6-oxohexanoate dehydrogenase, a 7-oxoheptanoate dehydrogenase, or amonooxygenase.

In some embodiments, the first terminal carboxyl group leading to thesynthesis of a C6 building block is enzymatically formed in hexanoyl-CoAby a thioesterase classified under EC 3.1.2.-, resulting in theproduction of hexanoate. The thioesterase can be the gene product ofYciA, tesB or Acot13 (Cantu et al., Protein Science, 2010, 19,1281-1295; Zhuang et al., Biochemistry, 2008, 47(9):2789-2796; Naggertet al., J. Biol. Chem., 1991, 266(17):11044-11050).

In some embodiments, the first terminal carboxyl group leading to thesynthesis of a C6 building block can be enzymatically formed in hexanalby an aldehyde dehydrogenase classified under EC 1.2.1.4 (see, Ho &Weiner, J. Bacteriol., 2005, 187(3):1067-1073), resulting in theproduction of hexanoate.

In some embodiments, the second terminal carboxyl group leading to thesynthesis of adipic acid can be enzymatically formed in adipatesemilaldehyde by an aldehyde dehydrogenase classified under EC 1.2.1.3(Guerrillot & Vandecasteele, Eur. J. Biochem., 1977, 81, 185-192).

In some embodiments, the second terminal carboxyl group leading to thesynthesis of adipic acid is enzymatically formed in adipate semialdehydeby a 6-oxohexanoate dehydrogenase such as the gene product of ChnE fromAcinetobacter sp. or 7-oxoheptanoate dehydrogenase such as the geneproduct of ThnG from Sphingomonas macrogolitabida (Iwaki et al., Appl.Environ. Microbiol., 1999, 65(11), 5158-5162; López-Sánchez et al.,Appl. Environ. Microbiol., 2010, 76(1), 110-118)). For example, a6-oxohexanoate dehydrogenase can be classified under EC 1.2.1.63. Forexample, a 7-oxoheptanoate dehydrogenase can be classified under EC1.2.1.-

In some embodiments, the second terminal carboxyl group leading to thesynthesis of adipic acid is enzymatically formed in adipate semialdehydeby a monooxygenase in the cytochrome P450 family such as CYP4F3B (see,e.g., Sanders et al., J. Lipid Research, 2005, 46(5):1001-1008; Sanderset al., The FASEB Journal, 2008, 22(6):2064-2071).

The utility of ω-oxidation in introducing carboxyl groups into alkaneshas been demonstrated in the yeast Candida tropicalis, leading to thesynthesis of adipic acid (Okuhara et al., Agr. Biol. Chem., 1971, 35(9),1376-1380).

Enzymes Generating the Terminal Amine Groups in the Biosynthesis of C6Building Blocks

As depicted in FIG. 5 and FIG. 6, terminal amine groups can beenzymatically formed using a ω-transaminase or a deacetylase.

In some embodiments, the first terminal amine group leading to thesynthesis of 6-aminohexanoic acid is enzymatically formed in adipatesemialdehyde by a ω-transaminase classified, for example, under EC2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 such asthat obtained from Chromobacterium violaceum (Genbank Accession No.AAQ59697.1, SEQ ID NO: 7), Pseudomonas aeruginosa (Genbank Accession No.AAG08191.1, SEQ ID NO: 8), Pseudomonas syringae (Genbank Accession No.AAY39893.1, SEQ ID NO: 9), Rhodobacter sphaeroides (Genbank AccessionNo. ABA81135.1, SEQ ID NO: 10), Vibrio Fluvialis (Genbank Accession No.AAA57874.1, SEQ ID NO: 11), Streptomyces griseus, or Clostridium viride.An additional ω-transaminase that can be used in the methods and hostsdescribed herein is from Escherichia coli (Genbank Accession No.AAA57874.1, SEQ ID NO: 11). Some of the ω-transaminases classified, forexample, under EC 2.6.1.29 or EC 2.6.1.82 are diamine ω-transaminases(e.g., SEQ ID NO:11).

The reversible ω-transaminase from Chromobacterium violaceum (GenbankAccession No. AAQ59697.1, SEQ ID NO: 7) has demonstrated analogousactivity accepting 6-aminohexanoic acid as amino donor, thus forming thefirst terminal amine group in adipate seminaldehyde (Kaulmann et al.,Enzyme and Microbial Technology, 2007, 41, 628-637).

The reversible 4-aminobubyrate:2-oxoglutarate transaminase fromStreptomyces griseus has demonstrated activity for the conversion of6-aminohexanoate to adipate semialdehyde (Yonaha et al., Eur. J.Biochem., 1985, 146, 101-106).

The reversible 5-aminovalerate transaminase from Clostridium viride hasdemonstrated activity for the conversion of 6-aminohexanoate to adipatesemialdehyde (Barker et al., J. Biol. Chem., 1987, 262(19), 8994-9003).

In some embodiments, the second terminal amine group leading to thesynthesis of hexamethylenediamine is enzymatically formed in6-aminohexanal by a diamine transaminase classified, for example, underEC 2.6.1.29 or classified, for example, under EC 2.6.1.82, such as thegene product of YgjG from E. coli (Genbank Accession No. AAA57874.1, SEQID NO: 12).

The gene product of ygjG accepts a broad range of diamine carbon chainlength substrates, such as putrescine, cadaverine and spermidine(Samsonova et al., BMC Microbiology, 2003, 3:2).

The diamine transaminase from E. coli strain B has demonstrated activityfor 1,7 diaminoheptane (Kim, The Journal of Chemistry, 1964, 239(3),783-786).

In some embodiments, the second terminal amine group leading to thesynthesis of heptamethylenediamine is enzymatically formed by adeacetylase classified, for example, under EC 3.5.1.62 such as anacetylputrescine deacetylase. The acetylputrescine deacetylase fromMicrococcus luteus K-11 accepts a broad range of carbon chain lengthsubstrates, such as acetylputrescine, acetylcadaverine andN⁸-acetylspermidine (see, for example, Suzuki et al., 1986, BBA—GeneralSubjects, 882(1):140-142).

Enzymes Generating the Terminal Hydroxyl Groups in the Biosynthesis ofC6 Building Blocks

As depicted in FIG. 7 and FIG. 8, the terminal hydroxyl group can beenzymatically forming using a monooxygenase or an alcohol dehydrogenase.

In some embodiments, the first terminal hydroxyl group leading to thesynthesis of C6 building blocks is enzymatically formed in hexanoate bya monooxygenase. For example, the monooxygenase CYP153A familyclassified, for example, under EC 1.14.15.1, is soluble and hasregio-specificity for terminal hydroxylation, accepting medium chainlength substrates (see, e.g., Koch et al., 2009, Appl. Environ.Microbiol., 75(2): 337-344; Funhoff et al., 2006, J. Bacteriol.,188(44): 5220-5227; Van Beilen & Funhoff, Current Opinion inBiotechnology, 2005, 16, 308-314; Nieder and Shapiro, J. Bacteriol.,1975, 122(1), 93-98). Although non-terminal hydroxylation is observed invitro for CYP153A, in vivo only 1-hydroxylation occurs (Funhoff et al.,J. Bacteriol., 2006, 188(14), 5220-5227).

The substrate specificity and activity of terminal monooxygenases hasbeen broadened via successfully, reducing the chain length specificityof CYP153A6 to below C8 (Koch et al., 2009, supra).

In some embodiments, the second terminal hydroxyl group leading to thesynthesis of 1,6 hexanediol is enzymatically formed in 6-hydroxyhexanalby an alcohol dehydrogenase classified under EC 1.1.1.- (e.g., EC1.1.1.1, 1.1.1.2, 1.1.1.21, or 1.1.1.184).

Biochemical Pathways

Pathways to Hexanoyl-CoA as Precursor Leading to Central Precursors toC6 Building Blocks

In some embodiments, hexanoyl-CoA is synthesized from the centralmetabolite, acetyl-CoA, by conversion of acetyl-CoA to acetoacetyl-CoAby an acetoacetyl-CoA thiolase classified, for example, under EC2.3.1.9, such as the gene product of atoB or phaA, or by an acetyl-CoAcarboxylase classified, for example, under EC 6.4.1.2 and anacetoacetyl-CoA synthase classified, for example, under EC 2.3.1.194 viamalonyl-CoA; followed by conversion of acetoacetyl-CoA to (S)3-hydroxybutanoyl-CoA by a 3-hydroxybutyryl-CoA dehydrogenaseclassified, for example, under EC 1.1.1.35, such as the gene product offadB or classified, for example, under EC 1.1.1.157 such as the geneproduct of hbd; followed by conversion of (S) 3-hydroxybutanoyl-CoA tocrotonyl-CoA by an enoyl-CoA hydratase classified, for example, under EC4.2.1.17 such as the gene product of crt; followed by conversion ofcrotonyl-CoA to butanoyl-CoA by a trans-2-enoyl-CoA reductaseclassified, for example, under EC 1.3.1.44 such as the gene product ofter or tdter; followed by conversion of butanoyl-CoA to3-oxo-hexanoyl-CoA by a β-ketothiolase classified, for example, under EC2.3.1.16 such as the gene product of bktB; followed by conversion of3-oxo-hexanoyl-CoA to (S) 3-hydroxyhexanoyl-CoA by a 3-hydroxyacyl-CoAdehydrogenase classified, for example, under EC 1.1.1.35 such as thegene product of fadB or classified, for example, under EC 1.1.1.157 suchas the gene product of hbd; followed by conversion of (S)3-hydroxyhexanoyl-CoA to hex-2-enoyl-CoA by an enoyl-CoA hydrataseclassified, for example, under EC 4.2.1.17 such as the gene product ofcrt; followed by conversion of hex-2-enoyl-CoA to hexanoyl-CoA by atrans-2-enoyl-CoA reductase classified, for example, under EC 1.3.1.44such as the gene product of ter or tdter. See FIG. 1.

In some embodiments, hexanoyl-CoA is synthesized from the centralmetabolite, acetyl-CoA, by conversion of acetyl-CoA to acetoacetyl-CoAby an acetoacetyl-CoA thiolase classified, for example, under EC2.3.1.9, such as the gene product of atoB or phaA, or by an acetyl-CoAcarboxylase classified, for example, under EC 6.4.1.2 & anacetoacetyl-CoA synthase classified, for example, under EC 2.3.1.194 viamalonyl-CoA; followed by conversion of acetoacetyl-CoA to (R)3-hydroxybutanoyl-CoA by a 3-acetoacetyl-CoA reductase classified, forexample, under EC 1.1.1.36 such as the gene product of phaB orclassified, for example, under EC 1.1.1.100, such as the gene product offabG; followed by conversion of (R) 3-hydroxybutanoyl-CoA tocrotonyl-CoA by an enoyl-CoA hydratase classified, for example, under EC4.2.1.119 such as the gene product of phaJ; followed by conversion ofcrotonyl-CoA to butanoyl-CoA by a trans-2-enoyl-CoA reductaseclassified, for example, under EC 1.3.1.38 or an acyl-dehydrogenaseclassified, for example, under EC 1.3.1.8; followed by conversion ofbutanoyl-CoA to 3-oxo-hexanoyl-CoA by a β-ketothiolase classified, forexample, under EC 2.3.1.16 such as the gene product of bktB; followed byconversion of 3-oxo-hexanoyl-CoA to (R) 3-hydroxyhexanoyl-CoA by a3-oxoacyl-CoA reductase classified, for example, under EC 1.1.1.36 suchas the gene product of phaB or classified, for example, under EC1.1.1.100 such as the gene product of fabG; followed by conversion of(R) 3-hydroxyhexanoyl-CoA to hex-2-enoyl-CoA by an enoyl-CoA hydrataseclassified, for example, under EC 4.2.1.119 such as the gene product ofphaJ; followed by conversion of hex-2-enoyl-CoA to hexanoyl-CoA by atrans-2-enoyl-CoA reductase classified, for example, under EC 1.3.1.38or an acyl-CoA dehydrogenase classified, for example, under EC 1.3.1.8.See FIG. 2.

Pathways Using Hexanoyl-CoA as Precursor Leading to the CentralPrecursor Hexanoate

In some embodiments, hexanoate is synthesized from the centralmetabolite, hexanoyl-CoA, by conversion of hexanoyl-CoA to hexanoate bya thioesterase classified, for example, under EC 3.1.2.- such as thegene product of YciA, tesB or Acot13.

In some embodiments, hexanoate is synthesized from the centralmetabolite, hexanoyl-CoA, by conversion of hexanoyl-CoA to hexanal by abutanal dehydrogenase classified, for example, under EC 1.2.1.57;followed by conversion of hexanal to hexanoate by an aldehydedehydrogenase classified, for example, under EC 1.2.1.4. See FIG. 3.

The conversion of hexanoyl-CoA to hexanal has been demonstrated for bothNADH and NADPH as co-factors (Palosaari and Rogers, J. Bacteriol., 1988,170(7), 2971-2976).

Pathways Using Hexanoate as Central Precursor to Adipic Acid

In some embodiments, adipic acid is synthesized from the centralprecursor, hexanoate, by conversion of hexanoate to 6-hydroxyhexanoateby a monooxygenase (e.g., cytochrome P450 such as from the CYP153 family(e.g., CYP153A6); followed by conversion of 6-hydroxyhexanoate toadipate semialdehyde by an alcohol dehydrogenase classified under EC1.1.1.- such as the gene product of YMR318C (classified, for example,under EC 1.1.1.2, see Genbank Accession No. CAA90836.1) (Larroy et al.,2002, Biochem J., 361(Pt 1), 163-172), cpnD (Iwaki et al., 2002, Appl.Environ. Microbiol., 68(11):5671-5684) or gabD (Lütke-Eversloh &Steinbüchel, 1999, FEMS Microbiology Letters, 181(1):63-71) or a6-hydroxyhexanoate dehydrogenase classified, for example, under EC1.1.1.258 such as the gene product of ChnD (Iwaki et al., Appl. Environ.Microbiol., 1999, 65(11):5158-5162); followed by conversion of adipatesemialdehyde to adipic acid by a dehydrogenase classified, for example,under EC 1.2.1.- such as a 7-oxoheptanoate dehydrogenase (e.g., the geneproduct of ThnG), a 6-oxohexanoate dehydrogenase (e.g., the gene productof ChnE), or an aldehyde dehydrogenase classified under EC 1.2.1.3. SeeFIG. 4. The alcohol dehydrogenase encoded by YMR318C has broad substratespecificity, including the oxidation of C6 alcohols.

In some embodiments, adipic acid is synthesized from the centralprecursor, hexanoate, by conversion of hexanoate to 6-hydroxyhexanoateby a monooxygenase (e.g., cytochrome P450) such as from the CYP153(e.g., CYP153A); followed by conversion of 6-hydroxyhexanoate to adipatesemialdehyde by a cytochrome P450 (Sanders et al., J. Lipid Research,2005, 46(5), 1001-1008; Sanders et al., The FASEB Journal, 2008, 22(6),2064-2071); followed by conversion of adipate semialdehyde to adipicacid by a monooxygenase in the cytochrome P450 family such as CYP4F3B.See FIG. 4.

Pathway Using Hexanoate as Central Precursor to 6-Aminohexanoate andε-Caprolactam

In some embodiments, 6-aminohexanoate is synthesized from the centralprecursor, hexanoate, by conversion of hexanoate to 6-hydroxyhexanoateby a monooxygenase e.g., cytochrome P450 such as from the CYP153 family(e.g., CYP153A6); followed by conversion of 6-hydroxyhexanoate toadipate semialdehyde by an alcohol dehydrogenase classified, forexample, under EC 1.1.1.2 such as the gene product of YMR318C, a6-hydroxyhexanoate dehydrogenase classified, for example, under EC1.1.1.258, a 5-hydroxypentanoate dehydrogenase classified, for example,under EC 1.1.1.- such as the gene product of cpnD, or a4-hydroxybutyrate dehydrogenase classified, for example, under EC1.1.1.- such as the gene product of gabD; followed by conversion ofadipate semialdehyde to 6-aminohexanoate by a ω-transaminase (EC2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 such asSEQ ID NOs:7-12, see above). See FIG. 5.

In some embodiments, ε-caprolactam is synthesized from the centralprecursor, hexanoate, by conversion of hexanoate to 6-hydroxyhexanoateby a monooxygenase such as from the CYP153 family (e.g., CYP153A);followed by conversion of 6-hydroxyhexanoate to adipate semialdehyde byan alcohol dehydrogenase classified, for example, under EC 1.1.1.2 suchas the gene product of YMR318C, a 6-hydroxyhexanoate dehydrogenaseclassified, for example, under EC 1.1.1.258, a 5-hydroxypentanoatedehydrogenase classified, for example, under EC 1.1.1.- such as the geneproduct of cpnD, or a 4-hydroxybutyrate dehydrogenase classified, forexample, under EC 1.1.1.- such as the gene product of gabD; followed byconversion of adipate semialdehyde to 6-aminohexanoate by aω-transaminase (EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, orEC 2.6.1.82); followed by conversion of 6-aminohexanoate toε-caprolactam by a hydrolase (EC 3.5.2.-). See FIG. 5.

Pathway Using 6-Aminohexanoate, 6-Hydroxyhexanoate, or AdipateSemialdehyde as a Central Precursor to Hexamethylenediamine

In some embodiments, hexamethylenediamine is synthesized from thecentral precursor, 6-aminohexanoate, by conversion of 6-aminohexanoateto 6-aminohexanal by a carboxylate reductase classified, for example,under EC 1.2.99.6 such as the gene product of car in combination with aphosphopantetheine transferase enhancer (e.g., encoded by a sfp genefrom Bacillus subtilis or npt gene from Nocardia) or the gene productsof GriC and GriD from Streptomyces griseus (Suzuki et al., J. Antibiot.,2007, 60(6), 380-387); followed by conversion of 6-aminohexanal tohexamethylenediamine by a ω-transaminase (e.g., EC 2.6.1.18, EC2.6.1.19, EC 2.6.1.48, EC 2.6.1.82 such as SEQ ID NOs:7-12). Thecarboxylate reductase can be obtained, for example, from Mycobacteriummarinum (Genbank Accession No. ACC40567.1, SEQ ID NO: 2), Mycobacteriumsmegmatis (Genbank Accession No. ABK71854.1, SEQ ID NO: 3), Segniliparusrugosus (Genbank Accession No. EFV 11917.1, SEQ ID NO: 4), Mycobacteriummassiliense (Genbank Accession No. EIV 11143.1, SEQ ID NO: 5), orSegniliparus rotundus (Genbank Accession No. ADG98140.1, SEQ ID NO: 6).See FIG. 6.

The carboxylate reductase encoded by the gene product of car andenhancer npt or sfp has broad substrate specificity, including terminaldifunctional C4 and C5 carboxylic acids (Venkitasubramanian et al.,Enzyme and Microbial Technology, 2008, 42, 130-137).

In some embodiments, hexamethylenediamine is synthesized from thecentral precursor, 6-hydroxyhexanoate (which can be produced asdescribed in FIG. 7), by conversion of 6-hydroxyhexanoate to6-hydroxyhexanal by a carboxylate reductase classified, for example,under EC 1.2.99.6 such as the gene product of car (see above) incombination with a phosphopantetheine transferase enhancer (e.g.,encoded by a sfp gene from Bacillus subtilis or npt gene from Nocardia)or the gene product of GriC & GriD (Suzuki et al., 2007, supra);followed by conversion of 6-aminohexanal to 6-aminohexanol by aω-transaminase classified, for example, under EC 2.6.1.18, EC 2.6.1.19,EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 such as SEQ ID NOs:7-12, seeabove; followed by conversion to 7-aminoheptanal by an alcoholdehydrogenase classified, for example, under EC 1.1.1.- (e.g., EC1.1.1.1, EC 1.1.1.2, EC 1.1.1.21, or EC 1.1.1.184) such as the geneproduct of YMR318C or YqhD (Liu et al., Microbiology, 2009, 155,2078-2085; Larroy et al., 2002, Biochem J., 361(Pt 1), 163-172; Jarboe,2011, Appl. Microbiol. Biotechnol., 89(2), 249-257) or the proteinhaving GenBank Accession No. CAA81612.1; followed by conversion toheptamethylenediamine by a ω-transaminase classified, for example, underEC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 suchas SEQ ID NOs:7-12, see above. See FIG. 6.

In some embodiments, hexamethylenediamine is synthesized from thecentral precursor, 6-aminohexanoate, by conversion of 6-aminohexanoateto N6-acetyl-6-aminohexanoate by an N-acetyltransferase such as a lysineN-acetyltransferase classified, for example, under EC 2.3.1.32; followedby conversion to N6-acetyl-6-aminohexanal by a carboxylate reductaseclassified, for example, under EC 1.2.99.6 such as the gene product ofcar (see above) in combination with a phosphopantetheine transferaseenhancer (e.g., encoded by a sfp gene from Bacillus subtilis or npt genefrom Nocardia) or the gene product of GriC & GriD; followed byconversion to N6-acetyl-1,6-diaminohexane by a ω-transaminaseclassified, for example, under EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC2.6.1.48, or EC 2.6.1.82 such as SEQ ID NOs:7-12, see above; followed byconversion to heptamethylenediamine by an acetylputrescine deacylaseclassified, for example, under EC 3.5.1.62. See, FIG. 6.

In some embodiments, hexamethylenediamine is synthesized from thecentral precursor, adipate semialdehyde, by conversion of adipatesemialdehyde to hexanedial by a carboxylate reductase classified, forexample, under EC 1.2.99.6 such as the gene product of car (see above)in combination with a phosphopantetheine transferase enhancer (e.g.,encoded by a sfp gene from Bacillus subtilis or npt gene from Nocardia)or the gene product of GriC & GriD; followed by conversion to6-aminohexanal by a ω-transaminase classified, for example, under EC2.6.1.18, EC 2.6.1.19, or EC 2.6.1.48; followed by conversion tohexamethylenediamine by a ω-transaminase classified, for example, underEC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 suchas SEQ ID NOs:7-12. See FIG. 6.

Pathways Using Hexanoate as Central Precursor to 6-Hydroxyhexanoate or1,6-Hexanediol

In some embodiments, 6-hydroxyhexanoate is synthesized from the centralprecursor, hexanoate, by conversion of hexanoate to 6-hydroxyhexanoateby a monooxygenase such as from the CYP153 family (e.g., CYP153A). SeeFIG. 7.

In some embodiments, 1,6 hexanediol is synthesized from the centralprecursor, 6-hydroxyhexanoate, by conversion of 6-hydroxyhexanoate to6-hydroxyhexanal by a carboxylate reductase classified, for example,under EC 1.2.99.6 such as the gene product of car (see above) incombination with a phosphopantetheine transferase enhancer (e.g.,encoded by a sfp gene from Bacillus subtilis or npt gene from Nocardia)or the gene products of GriC and GriD from Streptomyces griseus (Suzukiet al., J. Antibiot., 2007, 60(6), 380-387); followed by conversion of6-hydroxyhexanal to 1,6 hexanediol by an alcohol dehydrogenase(classified, for example, under EC 1.1.1.- such as EC 1.1.1.1, EC1.1.1.2, EC 1.1.1.21, or EC 1.1.1.184) such as the gene product ofYMR318C or YqhD (from E. coli, GenBank Accession No. AAA69178.1) (see,e.g., Liu et al., Microbiology, 2009, 155, 2078-2085; Larroy et al.,2002, Biochem J., 361(Pt 1), 163-172; or Jarboe, 2011, Appl. Microbiol.Biotechnol., 89(2), 249-257) or the protein having GenBank Accession No.CAA81612.1 (from Geobacillus stearothermophilus). See, FIG. 8.

Cultivation Strategy

In some embodiments, one or more C6 building blocks are biosynthesizedin a recombinant host using anaerobic, aerobic or micro-aerobiccultivation conditions. A non-cyclical or a cyclical cultivationstrategy can be used to achieve the desired cultivation conditions. Forexample, a non-cyclical strategy can be used to achieve anaerobic,aerobic or micro-aerobic cultivation conditions.

In some embodiments, a cyclical cultivation strategy can be used toalternate between anaerobic cultivation conditions and aerobiccultivation conditions.

In some embodiments, the cultivation strategy entails nutrientlimitation such as nitrogen, phosphate or oxygen limitation.

In some embodiments, a cell retention strategy using, for example,ceramic hollow fiber membranes can be employed to achieve and maintain ahigh cell density during either fed-batch or continuous fermentation.

In some embodiments, the principal carbon source fed to the fermentationin the synthesis of one or more C6 building blocks can derive frombiological or non-biological feedstocks.

In some embodiments, the biological feedstock can be or can derive from,monosaccharides, disaccharides, lignocellulose, hemicellulose,cellulose, lignin, levulinic acid and formic acid, triglycerides,glycerol, fatty acids, agricultural waste, condensed distillers'solubles, or municipal waste.

The efficient catabolism of crude glycerol stemming from the productionof biodiesel has been demonstrated in several microorganisms such asEscherichia coli, Cupriavidus necator, Pseudomonas oleavorans,Pseudomonas putida and Yarrowia lipolytica (Lee et al., Appl. Biochem.Biotechnol., 2012, 166:1801-1813; Yang et al., Biotechnology forBiofuels, 2012, 5:13; Meijnen et al., Appl. Microbiol. Biotechnol.,2011, 90:885-893).

The efficient catabolism of lignocellulosic-derived levulinic acid hasbeen demonstrated in several organisms such as Cupriavidus necator andPseudomonas putida in the synthesis of 3-hydroxyvalerate via theprecursor propanoyl-CoA (Jaremko and Yu, 2011, supra; Martin andPrather, J. Biotechnol., 2009, 139:61-67).

The efficient catabolism of lignin-derived aromatic compounds such asbenzoate analogues has been demonstrated in several microorganisms suchas Pseudomonas putida, Cupriavidus necator (Bugg et al., Current Opinionin Biotechnology, 2011, 22, 394-400; Pérez-Pantoja et al., FEMSMicrobiol. Rev., 2008, 32, 736-794).

The efficient utilization of agricultural waste, such as olive millwaste water has been demonstrated in several microorganisms, includingYarrowia lipolytica (Papanikolaou et al., Bioresour. Technol., 2008,99(7):2419-2428).

The efficient utilization of fermentable sugars such as monosaccharidesand disaccharides derived from cellulosic, hemicellulosic, cane and beetmolasses, cassava, corn and other agricultural sources has beendemonstrated for several microorganism such as Escherichia coli,Corynebacterium glutamicum and Lactobacillus delbrueckii and Lactococcuslactis (see, e.g., Hermann et al, J. Biotechnol., 2003, 104:155-172; Weeet al., Food Technol. Biotechnol., 2006, 44(2):163-172; Ohashi et al.,J. Bioscience and Bioengineering, 1999, 87(5):647-654).

The efficient utilization of furfural, derived from a variety ofagricultural lignocellulosic sources, has been demonstrated forCupriavidus necator (Li et al., Biodegradation, 2011, 22:1215-1225).

In some embodiments, the non-biological feedstock can be or can derivefrom natural gas, syngas, CO₂/H₂, methanol, ethanol, benzoate,non-volatile residue (NVR) or a caustic wash waste stream fromcyclohexane oxidation processes, or terephthalic acid/isophthalic acidmixture waste streams.

The efficient catabolism of methanol has been demonstrated for themethylotrophic yeast Pichia pastoris.

The efficient catabolism of ethanol has been demonstrated forClostridium kluyveri (Seedorf et al., Proc. Natl. Acad. Sci. USA, 2008,105(6) 2128-2133).

The efficient catabolism of CO₂ and H₂, which may be derived fromnatural gas and other chemical and petrochemical sources, has beendemonstrated for Cupriavidus necator (Prybylski et al., Energy,Sustainability and Society, 2012, 2:11).

The efficient catabolism of syngas has been demonstrated for numerousmicroorganisms, such as Clostridium ljungdahlii and Clostridiumautoethanogenum (Kopke et al., Applied and Environmental Microbiology,2011, 77(15):5467-5475).

The efficient catabolism of the non-volatile residue waste stream fromcyclohexane processes has been demonstrated for numerous microorganisms,such as Delftia acidovorans and Cupriavidus necator (Ramsay et al.,Applied and Environmental Microbiology, 1986, 52(1):152-156).

In some embodiments, the host microorganism is a prokaryote. Forexample, the prokaryote can be a bacterium from the genus Escherichiasuch as Escherichia coli; from the genus Clostridia such as Clostridiumljungdahlii, Clostridium autoethanogenum or Clostridium kluyveri; fromthe genus Corynebacteria such as Corynebacterium glutamicum; from thegenus Cupriavidus such as Cupriavidus necator or Cupriavidusmetallidurans; from the genus Pseudomonas such as Pseudomonasfluorescens, Pseudomonas putida or Pseudomonas oleavorans; from thegenus Delftia such as Delftia acidovorans; from the genus Bacillus suchas Bacillus subtillis; from the genus Lactobacillus such asLactobacillus delbrueckii; or from the genus Lactococcus such asLactococcus lactis. Such prokaryotes also can be a source of genes toconstruct recombinant host cells described herein that are capable ofproducing one or more C7 building blocks.

In some embodiments, the host microorganism is a eukaryote. For example,the eukaryote can be a filamentous fungus, e.g., one from the genusAspergillus such as Aspergillus niger. Alternatively, the eukaryote canbe a yeast, e.g., one from the genus Saccharomyces such as Saccharomycescerevisiae; from the genus Pichia such as Pichia pastoris; or from thegenus Yarrowia such as Yarrowia lipolytica; from the genus Issatchenkiasuch as Issathenkia orientalis; from the genus Debaryomyces such asDebaryomyces hansenii; from the genus Arxula such as Arxulaadenoinivorans; or from the genus Kluyveromyces such as Kluyveromyceslactis. Such eukaryotes also can be a source of genes to constructrecombinant host cells described herein that are capable of producingone or more C6 building blocks.

Metabolic Engineering

The present document provides methods involving less than all the stepsdescribed for all the above pathways. Such methods can involve, forexample, one, two, three, four, five, six, seven, eight, nine, ten,eleven, twelve or more of such steps. Where less than all the steps areincluded in such a method, the first, and in some embodiments the only,step can be any one of the steps listed.

Furthermore, recombinant hosts described herein can include anycombination of the above enzymes such that one or more of the steps,e.g., one, two, three, four, five, six, seven, eight, nine, ten, or moreof such steps, can be performed within a recombinant host. This documentprovides host cells of any of the genera and species listed andgenetically engineered to express one or more (e.g., two, three, four,five, six, seven, eight, nine, 10, 11, 12 or more) recombinant forms ofany of the enzymes recited in the document. Thus, for example, the hostcells can contain exogenous nucleic acids encoding enzymes catalyzingone or more of the steps of any of the pathways described herein.

In addition, this document recognizes that where enzymes have beendescribed as accepting CoA-activated substrates, analogous enzymeactivities associated with [acp]-bound substrates exist that are notnecessarily in the same enzyme class.

Also, this document recognizes that where enzymes have been describedaccepting (R)-enantiomers of substrate, analogous enzyme activitiesassociated with (S)-enantiomer substrates exist that are not necessarilyin the same enzyme class.

This document also recognizes that where an enzyme is shown to accept aparticular co-factor, such as NADPH, or co-substrate, such asacetyl-CoA, many enzymes are promiscuous in terms of accepting a numberof different co-factors or co-substrates in catalyzing a particularenzyme activity. Also, this document recognizes that where enzymes havehigh specificity for e.g., a particular co-factor such as NADH, anenzyme with similar or identical activity that has high specificity forthe co-factor NADPH may be in a different enzyme class.

In some embodiments, the enzymes in the pathways outlined herein are theresult of enzyme engineering via non-direct or rational enzyme designapproaches with aims of improving activity, improving specificity,reducing feedback inhibition, reducing repression, improving enzymesolubility, changing stereo-specificity, or changing co-factorspecificity.

In some embodiments, the enzymes in the pathways outlined here can begene dosed, i.e., overexpressed, into the resulting genetically modifiedorganism via episomal or chromosomal integration approaches.

In some embodiments, genome-scale system biology techniques such as FluxBalance Analysis can be utilized to devise genome scale attenuation orknockout strategies for directing carbon flux to a C6 building block.

Attenuation strategies include, but are not limited to; the use oftransposons, homologous recombination (double cross-over approach),mutagenesis, enzyme inhibitors and RNAi interference.

In some embodiments, fluxomic, metabolomic and transcriptomal data canbe utilized to inform or support genome-scale system biology techniques,thereby devising genome scale attenuation or knockout strategies indirecting carbon flux to a C6 building block.

In some embodiments, the host microorganism's tolerance to highconcentrations of a C6 building block can be improved through continuouscultivation in a selective environment.

In some embodiments, the host microorganism's endogenous biochemicalnetwork can be attenuated or augmented to (1) ensure the intracellularavailability of acetyl-CoA, (2) create an NADH or NADPH imbalance thatmay only be balanced via the formation of one or more C6 buildingblocks, (3) prevent degradation of central metabolites, centralprecursors leading to and including one or more C6 building blocksand/or (4) ensure efficient efflux from the cell.

In some embodiments requiring intracellular availability of acetyl-CoAfor C6 building block synthesis, endogenous enzymes catalyzing thehydrolysis of acetyl-CoA such as short-chain length thioesterases can beattenuated in the host organism.

In some embodiments requiring the intracellular availability ofacetyl-CoA for C6 building block synthesis, an endogenousphosphotransacetylase generating acetate such as pta can be attenuated(Shen et al., Appl. Environ. Microbiol., 2011, 77(9):2905-2915).

In some embodiments requiring the intracellular availability ofacetyl-CoA for C6 building block synthesis, an endogenous gene in anacetate synthesis pathway encoding an acetate kinase, such as ack, canbe attenuated.

In some embodiments requiring the intracellular availability ofacetyl-CoA and NADH for C6 building block synthesis, an endogenous geneencoding an enzyme that catalyzes the degradation of pyruvate to lactatesuch as lactate dehydrogenase encoded by ldhA can be attenuated (Shen etal., 2011, supra).

In some embodiments requiring the intracellular availability ofacetyl-CoA and NADH for C6 building block synthesis, endogenous genesencoding enzymes, such as menaquinol-fumarate oxidoreductase, thatcatalyze the degradation of phophoenolpyruvate to succinate such asfrdBC can be attenuated (see, e.g., Shen et al., 2011, supra).

In some embodiments requiring the intracellular availability ofacetyl-CoA and NADH for C6 building block synthesis, an endogenous geneencoding an enzyme that catalyzes the degradation of acetyl-CoA toethanol such as the alcohol dehydrogenase encoded by adhE can beattenuated (Shen et al., 2011, supra).

In some embodiments, where pathways require excess NADH co-factor for C6building block synthesis, a recombinant formate dehydrogenase gene canbe overexpressed in the host organism (Shen et al., 2011, supra).

In some embodiments, where pathways require excess NADH co-factor for C7building block synthesis, a recombinant NADH-consuming transhydrogenasecan be attenuated.

In some embodiments, an endogenous gene encoding an enzyme thatcatalyzes the degradation of pyruvate to ethanol such as pyruvatedecarboxylase can be attenuated.

In some embodiments, an endogenous gene encoding an enzyme thatcatalyzes the generation of isobutanol such as a 2-oxoacid decarboxylasecan be attenuated.

In some embodiments requiring the intracellular availability ofacetyl-CoA for C6 building block synthesis, a recombinant acetyl-CoAsynthetase such as the gene product of acs can be overexpressed in themicroorganism (Satoh et al., J. Bioscience and Bioengineering, 2003,95(4):335-341).

In some embodiments, carbon flux can be directed into the pentosephosphate cycle to increase the supply of NADPH by attenuating anendogenous glucose-6-phosphate isomerase (EC 5.3.1.9).

In some embodiments, carbon flux can be redirected into the pentosephosphate cycle to increase the supply of NADPH by overexpression a6-phosphogluconate dehydrogenase and/or a transketolase (Lee et al.,2003, Biotechnology Progress, 19(5), 1444-1449).

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of a C6 building block, a gene such as UdhA encoding apuridine nucleotide transhydrogenase can be overexpressed in the hostorganisms (Brigham et al., Advanced Biofuels and Bioproducts, 2012,Chapter 39, 1065-1090).

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of a C6 Building Block, a recombinantglyceraldehyde-3-phosphate-dehydrogenase gene such as GapN can beoverexpressed in the host organisms (Brigham et al., 2012, supra).

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of a C6 building block, a recombinant malic enzyme genesuch as macA or maeB can be overexpressed in the host organisms (Brighamet al., 2012, supra).

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of a C6 building block, a recombinant glucose-6-phosphatedehydrogenase gene such as zwf can be overexpressed in the hostorganisms (Lim et al., J. Bioscience and Bioengineering, 2002, 93(6),543-549).

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of a C6 building block, a recombinant fructose 1,6diphosphatase gene such as fbp can be overexpressed in the hostorganisms (Becker et al., J. Biotechnol., 2007, 132:99-109).

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of a C6 building block, endogenous triose phosphateisomerase (EC 5.3.1.1) can be attenuated.

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of a C6 building block, a recombinant glucosedehydrogenase such as the gene product of gdh can be overexpressed inthe host organism (Satoh et al., J. Bioscience and Bioengineering, 2003,95(4):335-341).

In some embodiments, endogenous enzymes facilitating the conversion ofNADPH to NADH can be attenuated, such as the NADH generation cycle thatmay be generated via inter-conversion of glutamate dehydrogenasesclassified under EC 1.4.1.2 (NADH-specific) and EC 1.4.1.4(NADPH-specific).

In some embodiments, an endogenous glutamate dehydrogenase (EC 1.4.1.3)that utilizes both NADH and NADPH as co-factors can be attenuated.

In some embodiments, a membrane-bound cytochrome P450 such as CYP4F3Bcan be solubilized by only expressing the cytosolic domain and not theN-terminal region that anchors the P450 to the endoplasmic reticulum(Scheller et al., J. Biol. Chem., 1994, 269(17):12779-12783).

In some embodiments, an enoyl-CoA reductase can be solubilized viaexpression as a fusion protein with a small soluble protein, forexample, the maltose binding protein (Gloerich et al., FEBS Letters,2006, 580, 2092-2096).

In some embodiments using hosts that naturally accumulatepolyhydroxyalkanoates, the endogenous polymer synthase enzymes can beattenuated in the host strain.

In some embodiments, a L-alanine dehydrogenase can be overexpressed inthe host to regenerate L-alanine from pyruvate as an amino donor forω-transaminase reactions.

In some embodiments, a L-glutamate dehydrogenase, a L-glutaminesynthetase, or a glutamate synthase can be overexpressed in the host toregenerate L-glutamate from 2-oxoglutarate as an amino donor forω-transaminase reactions.

In some embodiments, enzymes such as a pimeloyl-CoA dehydrogenaseclassified under, EC 1.3.1.62; an acyl-CoA dehydrogenase classified, forexample, under EC 1.3.8.7, EC 1.3.8.1, or EC 1.3.99.-; and/or abutyryl-CoA dehydrogenase classified, for example, under EC 1.3.8.6 thatdegrade central metabolites and central precursors leading to andincluding C6 building blocks can be attenuated.

In some embodiments, endogenous enzymes activating C6 building blocksvia Coenzyme A esterification such as CoA-ligases (e.g., an adipyl-CoAsynthetase) classified under, for example, EC 6.2.1.- can be attenuated.

In some embodiments, the efflux of a C6 building block across the cellmembrane to the extracellular media can be enhanced or amplified bygenetically engineering structural modifications to the cell membrane orincreasing any associated transporter activity for a C6 building block.

The efflux of hexamethylenediamine can be enhanced or amplified byoverexpressing broad substrate range multidrug transporters such as Bltfrom Bacillus subtilis (Woolridge et al., 1997, J. Biol. Chem.,272(14):8864-8866); AcrB and AcrD from Escherichia coli (Elkins &Nikaido, 2002, J. Bacteriol., 184(23), 6490-6499), NorA fromStaphylococcus aereus (Ng et al., 1994, Antimicrob Agents Chemother,38(6), 1345-1355), or Bmr from Bacillus subtilis (Neyfakh, 1992,Antimicrob Agents Chemother, 36(2), 484-485).

The efflux of 6-aminohexanoate and heptamethylenediamine can be enhancedor amplified by overexpressing the solute transporters such as the lysEtransporter from Corynebacterium glutamicum (Bellmann et al., 2001,Microbiology, 147, 1765-1774).

The efflux of adipic acid can be enhanced or amplified by overexpressinga dicarboxylate transporter such as the SucE transporter fromCorynebacterium glutamicum (Huhn et al., Appl. Microbiol. & Biotech.,89(2), 327-335).

Producing C6 Building Blocks Using a Recombinant Host

Typically, one or more C6 building blocks can be produced by providing ahost microorganism and culturing the provided microorganism with aculture medium containing a suitable carbon source as described above.In general, the culture media and/or culture conditions can be such thatthe microorganisms grow to an adequate density and produce a C6 buildingblock efficiently. For large-scale production processes, any method canbe used such as those described elsewhere (Manual of IndustrialMicrobiology and Biotechnology, 2^(nd) Edition, Editors: A. L. Demainand J. E. Davies, ASM Press; and Principles of Fermentation Technology,P. F. Stanbury and A. Whitaker, Pergamon). Briefly, a large tank (e.g.,a 100 gallon, 200 gallon, 500 gallon, or more tank) containing anappropriate culture medium is inoculated with a particularmicroorganism. After inoculation, the microorganism is incubated toallow biomass to be produced. Once a desired biomass is reached, thebroth containing the microorganisms can be transferred to a second tank.This second tank can be any size. For example, the second tank can belarger, smaller, or the same size as the first tank. Typically, thesecond tank is larger than the first such that additional culture mediumcan be added to the broth from the first tank. In addition, the culturemedium within this second tank can be the same as, or different from,that used in the first tank.

Once transferred, the microorganisms can be incubated to allow for theproduction of a C6 building block. Once produced, any method can be usedto isolate C6 building blocks. For example, C6 building blocks can berecovered selectively from the fermentation broth via adsorptionprocesses. In the case of adipic acid and 6-aminoheptanoic acid, theresulting eluate can be further concentrated via evaporation,crystallized via evaporative and/or cooling crystallization, and thecrystals recovered via centrifugation. In the case ofhexamethylenediamine and 1,6-heptanediol, distillation may be employedto achieve the desired product purity.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Genome-Scale Attenuation Strategy for CyclicalSynthesis of Adipic Acid from Glucose in Saccharomyces cerevisiae

Genome-scale Flux Balance Analysis was undertaken using the genome-scalemodel for Saccharomyces cerevisiae designated iMM904 (Mo et al., BMCSystems Biology, 2009, 3(37), 1-17).

The IMM904 model was extended by including ω-oxidation pathways asoutlined in published work for eukaryotic organisms (Sanders et al.,Journal of Lipid Research, 2005, 46(5), 1001-1008). Furthermore, theβ-oxidation reactions in the peroxisome of the iMM904 model wereextended and relevant membrane transport reactions were included. Theinactivation of a fumarate reductase was required during validation ofthe extended model to align model fluxes with experimental flux data.

The NADH-specific enzymatic reactions outlined in FIG. 1 wereincorporated into the model.

Allowance was made for the membrane transport of hexanoic acid andadipic acid to and from the extracellular media.

The stoichiometric balance of NADH in the production of 1-butanol in E.coli is far more efficient when overexpressing formate dehydrogenase(Shen et al., Appl. Environ. Microbiol., 2011, 77(9), 2905-2915). TheiMM904 model includes formate dehydrogenase, but lacked pyruvate formatelyase activity, which was included into the Saccharomyces cerevisiaemodel.

The co-factor specificity of NAD(H) or NADP(H) dependent enzymes wasassessed and where published literature was not unequivocal in terms ofspecificity, a promiscuous enzyme was assumed.

The metabolic engineering workbench, Optflux, was used to search thesolution space associated with the biochemical network for attenuationstrategies that (1) produce hexanoate anaerobically from glucose,followed by (2) production of adipate aerobically from the extracellularhexanoate, whilst co-feeding glucose as carbon and energy source.

The optimization trials found four beneficial attenuations; viz. (1)attenuating glucose-6-phosphate isomerase, directing flux into thepentose phosphate cycle; (2) attenuating pyruvate decarboxylase oralcohol dehydrogenase, preventing ethanol production; (3) attenuating2-oxoacid decarboxylase, preventing isobutanol production and (4)inactivating β-oxidation, preventing central precursor, centralmetabolite and adipic acid degradation.

The attenuations in this S. cerevisiae mutant using glucose as carbonand energy source, favored the balancing of NADH via the production ofhexanoic acid as a means of maximizing biomass growth.

Overexpression of formate dehydrogenase in the S. cerevisiae mutanteliminated the by-product formation of formate and pyruvate, producinghexanoate with a molar yield of 0.62 [(mol hexanoate)/(mol glucose)].

Cycling from anaerobic to aerobic cultivation, while maintaining aglucose feed rate to match the growth rate under anaerobic conditions,resulted in an overall molar yield of 0.38 [(mol adipate)/(mol totalglucose)].

In-silico attenuation of the biochemical network using a validated modeldetermined that a cultivation strategy, cycling between anaerobic andaerobic conditions, produces predominantly adipic acid from the fedglucose.

Example 2 Genome-Scale Attenuation Strategy for Micro-Aerobic Synthesisfrom Glucose Using NADH Imbalance to Direct Carbon Flux Towards AdipicAcid in Saccharomyces cerevisiae

The iMM904 model outlined in Example 1 was utilized to assess thenon-cyclical production of adipic acid using glucose as carbon andenergy source under micro-aerobic, substrate oxidation and growthlimiting cultivation conditions.

Using the extended iMM904 model and the metabolic engineering workbench,Optflux; optimization trials found an optimal attenuation strategyincluding: (1) attenuating hexanoate transport to the extracellularmedia; (2) attenuating ethanol excretion to the extracellular media; (3)attenuating 2-hydroxybutyrate oxidoreductase, preventing2-hydroxybutyrate production; (4) attenuating DL glycerol-3-phosphatase,preventing glycerol production and (5) attenuating malate dehydrogenase,preventing inter-conversion of NADH to NADPH.

The resulting S. cerevisiae mutant produces adipate as the mostadvantageous means of balancing NADH to maximize biomass growth,producing adipate with a molar yield of 0.71 [(mol adipate)/(molglucose)].

In-silico attenuation of the biochemical network using a validated modeldetermined that a non-cyclical cultivation strategy under micro-aerobicconditions produces predominantly adipic acid from the fed glucose.

Example 3 Genome-Scale Attenuation Strategy for Aerobic Synthesis fromGlucose Using NADPH Imbalance to Direct Carbon Flux Towards Adipic Acidin Saccharomyces cerevisiae

The iMM904 model outlined in Example 1 was utilized to assess thenon-cyclical production of adipic acid using glucose as carbon andenergy source under aerobic cultivation and growth limiting conditions.

The NADH-specific enzymatic reactions outlined in FIG. 1 were replacedby the equivalent NADPH-specific enzymatic reactions outlined in FIG. 2.

Using the extended iMM904 model and the metabolic engineering workbench,Optflux; optimization trials found an optimal attenuation strategyincluding; (1) attenuating triose phosphate isomerase/phosphoglucoseisomerase, redirecting flux into the pentose phosphate cycle; (2)preventing the inter-conversion of NADPH to NADH, by attenuating theNADH-dependent glutamate dehydrogenase and proline oxidase.

The resulting S. cerevisiae mutant produces adipate as the mostadvantageous means of balancing NADPH to maximize biomass growth,producing adipate with a molar yield of 0.4 [(mol adipate)/(molglucose)].

In-silico attenuation of the biochemical network using a validated modeldetermined that a non-cyclical cultivation strategy under aerobicconditions produces predominantly adipic acid from the fed glucose.

Example 4 Enzyme Activity of Thioesterases Using Hexanoyl-CoA as aSubstrate and Forming Hexanoate

A nucleotide sequence encoding a His tag was added to the tesB gene fromEscherichia coli that encodes a thioesterase (SEQ ID NO 1, see FIG. 9),such that an N-terminal HIS tagged thioesterase could be produced. Themodified tesB gene was cloned into a pET15b expression vector undercontrol of the T7 promoter. The expression vector was transformed into aBL21[DE3] E. coli host. The resulting recombinant E. coli strain wascultivated at 37° C. in a 500 mL shake flask culture containing 50 mLLuria Broth (LB) media and antibiotic selection pressure, with shakingat 230 rpm. The culture was induced overnight at 17° C. using 0.5 mMIPTG.

The pellet from the induced shake flask culture was harvested viacentrifugation. The pellet was resuspended and lysed in Y-per™ solution(ThermoScientific, Rockford, Ill.). The cell debris was separated fromthe supernatant via centrifugation. The thioesterase was purified fromthe supernatant using Ni-affinity chromatography and the eluate wasbuffer exchanged and concentrated via ultrafiltration.

The enzyme activity assay was performed in triplicate in a buffercomposed of 50 mM phosphate buffer (pH=7.4), 0.1 mM Ellman's reagent,and 133 μM of hexanoyl-CoA (as substrate). The enzyme activity assayreaction was initiated by adding 0.8 μM of the tesB gene product to theassay buffer containing the hexanoyl-CoA and incubating at 37° C. for 20min. The release of Coenzyme A was monitored by absorbance at 412 nm.The absorbance associated with the substrate only control, whichcontained boiled enzyme, was subtracted from the active enzyme assayabsorbance and compared to the empty vector control. The gene product oftesB accepted hexanoyl-CoA as substrate as confirmed via relativespectrophotometry (see FIG. 10) and synthesized hexanoate as a reactionproduct.

Example 5 Enzyme Activity of ω-Transaminase Using Adipate Semialdehydeas Substrate and Forming 6-Aminohexanoate

A nucleotide sequence encoding a His-tag was added to the genes fromChromobacterium violaceum, Pseudomonas aeruginosa, Pseudomonas syringae,Rhodobacter sphaeroides, and Vibrio fluvialis encoding theω-transaminases of SEQ ID NOs: 7, 8, 9, 10 and 12, respectively (seeFIG. 9) such that N-terminal HIS tagged CD-transaminases could beproduced. Each of the resulting modified genes was cloned into a pET21aexpression vector under control of the T7 promoter and each expressionvector was transformed into a BL21[DE3] E. coli host. The resultingrecombinant E. coli strains were cultivated at 37° C. in a 250 mL shakeflask culture containing 50 mL LB media and antibiotic selectionpressure, with shaking at 230 rpm. Each culture was induced overnight at16° C. using 1 mM IPTG.

The pellet from each induced shake flask culture was harvested viacentrifugation. Each pellet was resuspended and lysed via sonication.The cell debris was separated from the supernatant via centrifugationand the cell free extract was used immediately in enzyme activityassays.

Enzyme activity assays in the reverse direction (i.e., 6-aminohexanoateto adipate semialdehyde) were performed in a buffer composed of a finalconcentration of 50 mM HEPES buffer (pH=7.5), 10 mM 6-aminohexanoate, 10mM pyruvate and 100 μM pyridoxyl 5′ phosphate. Each enzyme activityassay reaction was initiated by adding cell free extract of theω-transaminase gene product or the empty vector control to the assaybuffer containing the 6-aminohexanoate and incubated at 25° C. for 24 h,with shaking at 250 rpm. The formation of L-alanine from pyruvate wasquantified via RP-HPLC.

Each enzyme only control without 6-aminoheptanoate demonstrated low baseline conversion of pyruvate to L-alanine See FIG. 16. The gene productof SEQ ID NO 7, SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 12 accepted6-aminohexanote as substrate as confirmed against the empty vectorcontrol. See FIG. 17.

Enzyme activity in the forward direction (i.e., adipate semialdehyde to6-aminohexanoate) was confirmed for the transaminases of SEQ ID NO 7,SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 12. Enzyme activityassays were performed in a buffer composed of a final concentration of50 mM HEPES buffer (pH=7.5), 10 mM adipate semialdehyde, 10 mM L-alanineand 100 μM pyridoxyl 5′ phosphate. Each enzyme activity assay reactionwas initiated by adding a cell free extract of the ω-transaminase geneproduct or the empty vector control to the assay buffer containing theadipate semialdehyde and incubated at 25° C. for 4 h, with shaking at250 rpm. The formation of pyruvate was quantified via RP-HPLC.

The gene product of SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10and SEQ ID NO 12 accepted adipate semialdehyde as substrate as confirmedagainst the empty vector control. See FIG. 18. The reversibility of theω-transaminase activity was confirmed, demonstrating that theω-transaminases of SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10and SEQ ID NO 12 accepted adipate semialdehyde as substrate andsynthesized 6-aminohexanoate as a reaction product.

Example 6 Enzyme Activity of Carboxylate Reductase Using Adipate asSubstrate and Forming Adipate Semialdehyde

A nucleotide sequence encoding a HIS-tag was added to the genes fromSegniliparus rugosus and Segniliparus rotundus that encode thecarboxylate reductases of SEQ ID NOs: 4 and 6, respectively (see FIG.9), such that N-terminal HIS tagged carboxylate reductases could beproduced. Each of the modified genes was cloned into a pET Duetexpression vector along with a sfp gene encoding a HIS-taggedphosphopantetheine transferase from Bacillus subtilis, both under the T7promoter. Each expression vector was transformed into a BL21[DE3] E.coli host and the resulting recombinant E. coli strains were cultivatedat 37° C. in a 250 mL shake flask culture containing 50 mL LB media andantibiotic selection pressure, with shaking at 230 rpm. Each culture wasinduced overnight at 37° C. using an auto-induction media.

The pellet from each induced shake flask culture was harvested viacentrifugation. Each pellet was resuspended and lysed via sonication,and the cell debris was separated from the supernatant viacentrifugation. The carboxylate reductases and phosphopantetheinetransferases were purified from the supernatant using Ni-affinitychromatography, diluted 10-fold into 50 mM HEPES buffer (pH=7.5), andconcentrated via ultrafiltration.

Enzyme activity assays (i.e., from adipate to adipate semialdehyde) wereperformed in triplicate in a buffer composed of a final concentration of50 mM HEPES buffer (pH=7.5), 2 mM adipate, 10 mM MgCl₂, 1 mM ATP and 1mM NADPH. Each enzyme activity assay reaction was initiated by addingpurified carboxylate reductase and phosphopantetheine transferase geneproducts or the empty vector control to the assay buffer containing theadipate and then incubated at room temperature for 20 min. Theconsumption of NADPH was monitored by absorbance at 340 nm. Each enzymeonly control without adipate demonstrated low base line consumption ofNADPH. See FIG. 11.

The gene products of SEQ ID NO 4 and SEQ ID NO 6, enhanced by the geneproduct of sfp, accepted adipate as substrate, as confirmed against theempty vector control (see FIG. 12), and synthesized adipatesemialdehyde.

Example 7 Enzyme Activity of Carboxylate Reductase Using6-Hydroxyhexanoate as Substrate and Forming 6-Hydroxyhexanal

A nucleotide sequence encoding a His-tag was added to the genes fromMycobacterium marinum, Mycobacterium smegmatis, Mycobacterium smegmatis,Segniliparus rugosus, Mycobacterium massiliense, and Segniliparusrotundus that encode the carboxylate reductases of SEQ ID NOs: 2-6,respectively (see FIG. 9) such that N-terminal HIS tagged carboxylatereductases could be produced. Each of the modified genes was cloned intoa pET Duet expression vector alongside a sfp gene encoding a His-taggedphosphopantetheine transferase from Bacillus subtilis, both undercontrol of the T7 promoter. Each expression vector was transformed intoa BL21[DE3] E. coli host along with the expression vectors from Example3. Each resulting recombinant E. coli strain was cultivated at 37° C. ina 250 mL shake flask culture containing 50 mL LB media and antibioticselection pressure, with shaking at 230 rpm. Each culture was inducedovernight at 37° C. using an auto-induction media.

The pellet from each induced shake flask culture was harvested viacentrifugation. Each pellet was resuspended and lysed via sonication.The cell debris was separated from the supernatant via centrifugation.The carboxylate reductases and phosphopantetheine transferase werepurified from the supernatant using Ni-affinity chromatography, diluted10-fold into 50 mM HEPES buffer (pH=7.5) and concentrated viaultrafiltration.

Enzyme activity (i.e., 6-hydroxyhexanoate to 6-hydroxyhexanal) assayswere performed in triplicate in a buffer composed of a finalconcentration of 50 mM HEPES buffer (pH=7.5), 2 mM 6-hydroxyhexanal, 10mM MgCl₂, 1 mM ATP, and 1 mM NADPH. Each enzyme activity assay reactionwas initiated by adding purified carboxylate reductase andphosphopantetheine transferase or the empty vector control to the assaybuffer containing the 6-hydroxyhexanoate and then incubated at roomtemperature for 20 min. The consumption of NADPH was monitored byabsorbance at 340 nm. Each enzyme only control without6-hydroxyhexanoate demonstrated low base line consumption of NADPH. SeeFIG. 11.

The gene products of SEQ ID NO 2-6, enhanced by the gene product of sfp,accepted 6-hydroxyhexanoate as substrate as confirmed against the emptyvector control (see FIG. 13), and synthesized 6-hydroxyhexanal.

Example 8 Enzyme Activity of ω-Transaminase for 6-Aminohexanol, Forming6-Oxohexanol

A nucleotide sequence encoding an N-terminal His-tag was added to theChromobacterium violaceum, Pseudomonas aeruginosa, Pseudomonas syringae,Rhodobacter sphaeroides, Escherichia coli, and Vibrio fluvialis genesencoding the w-transaminases of SEQ ID NOs: 7-12, respectively (see FIG.9) such that N-terminal HIS tagged ω-transaminases could be produced.The modified genes were cloned into a pET21a expression vector under theT7 promoter. Each expression vector was transformed into a BL21[DE3] E.coli host. Each resulting recombinant E. coli strain were cultivated at37° C. in a 250 mL shake flask culture containing 50 mL LB media andantibiotic selection pressure, with shaking at 230 rpm. Each culture wasinduced overnight at 16° C. using 1 mM IPTG.

The pellet from each induced shake flask culture was harvested viacentrifugation. Each pellet was resuspended and lysed via sonication.The cell debris was separated from the supernatant via centrifugationand the cell free extract was used immediately in enzyme activityassays.

Enzyme activity assays in the reverse direction (i.e., 6-aminohexanol to6-oxohexanol) were performed in a buffer composed of a finalconcentration of 50 mM HEPES buffer (pH=7.5), 10 mM 6-aminohexanol, 10mM pyruvate, and 100 μM pyridoxyl 5′ phosphate. Each enzyme activityassay reaction was initiated by adding cell free extract of theω-transaminase gene product or the empty vector control to the assaybuffer containing the 6-aminohexanol and then incubated at 25° C. for 4h, with shaking at 250 rpm. The formation of L-alanine was quantifiedvia RP-HPLC.

Each enzyme only control without 6-aminohexanol had low base lineconversion of pyruvate to L-alanine See FIG. 16.

The gene products of SEQ ID NOs: 7-12 accepted 6-aminohexanol assubstrate as confirmed against the empty vector control (see FIG. 21)and synthesized 6-oxohexanol as reaction product. Given thereversibility of the ω-transaminase activity (see Example 5), it can beconcluded that the gene products of SEQ ID NOs: 7-12 accept6-aminohexanol as substrate and form 6-oxohexanol.

Example 9 Enzyme Activity of ω-Transaminase Using Hexamethylenediamineas Substrate and Forming 6-Aminohexanal

A nucleotide sequence encoding an N-terminal His-tag was added to theChromobacterium violaceum, Pseudomonas aeruginosa, Pseudomonas syringae,Rhodobacter sphaeroides, Escherichia coli, and Vibrio fluvialis genesencoding the w-transaminases of SEQ ID NOs: 7-12, respectively (see FIG.9) such that N-terminal HIS tagged ω-transaminases could be produced.The modified genes were cloned into a pET21a expression vector under theT7 promoter. Each expression vector was transformed into a BL21[DE3] E.coli host. Each resulting recombinant E. coli strain were cultivated at37° C. in a 250 mL shake flask culture containing 50 mL LB media andantibiotic selection pressure, with shaking at 230 rpm. Each culture wasinduced overnight at 16° C. using 1 mM IPTG.

The pellet from each induced shake flask culture was harvested viacentrifugation. Each pellet was resuspended and lysed via sonication.The cell debris was separated from the supernatant via centrifugationand the cell free extract was used immediately in enzyme activityassays.

Enzyme activity assays in the reverse direction (i.e.,hexamethylenediamine to 6-aminohexanal) were performed in a buffercomposed of a final concentration of 50 mM HEPES buffer (pH=7.5), 10 mMhexamethylenediamine, 10 mM pyruvate, and 100 μM pyridoxyl 5′ phosphate.Each enzyme activity assay reaction was initiated by adding cell freeextract of the ω-transaminase gene product or the empty vector controlto the assay buffer containing the hexamethylenediamine and thenincubated at 25° C. for 4 h, with shaking at 250 rpm. The formation ofL-alanine was quantified via RP-HPLC.

Each enzyme only control without hexamethylenediamine had low base lineconversion of pyruvate to L-alanine See FIG. 16.

The gene products of SEQ ID NO 7-12 accepted hexamethylenediamine assubstrate as confirmed against the empty vector control (see FIG. 19)and synthesized 6-aminohexanal as reaction product. Given thereversibility of the ω-transaminase activity (see Example 5), it can beconcluded that the gene products of SEQ ID NOs: 7-12 accept6-aminohexanal as substrate and form hexamethylenediamine.

Example 10 Enzyme Activity of Carboxylate Reductase forN6-Acetyl-6-Aminohexanoate, Forming N6-Acetyl-6-Aminohexanal

The activity of each of the N-terminal His-tagged carboxylate reductasesof SEQ ID NOs: 4-6 (see Example 7, and FIG. 9) for convertingN6-acetyl-6-aminohexanoate to N6-acetyl-6-aminohexanal was assayed intriplicate in a buffer composed of a final concentration of 50 mM HEPESbuffer (pH=7.5), 2 mM N6-acetyl-6-aminohexanoate, 10 mM MgCl₂, 1 mM ATP,and 1 mM NADPH. The assays were initiated by adding purified carboxylatereductase and phosphopantetheine transferase or the empty vector controlto the assay buffer containing the N6-acetyl-6-aminohexanoate thenincubated at room temperature for 20 min. The consumption of NADPH wasmonitored by absorbance at 340 nm. Each enzyme only control withoutN6-acetyl-6-aminohexanoate demonstrated low base line consumption ofNADPH. See FIG. 11.

The gene products of SEQ ID NO 4-6, enhanced by the gene product of sfp,accepted N6-acetyl-6-aminohexanoate as substrate as confirmed againstthe empty vector control (see FIG. 14), and synthesizedN6-acetyl-6-aminohexanal.

Example 11 Enzyme Activity of ω-Transaminase UsingN6-Acetyl-1,6-Diaminohexane, and Forming N6-Acetyl-6-Aminohexanal

The activity of the N-terminal His-tagged ω-transaminases of SEQ ID NOs:7-12 (see Example 9, and FIG. 9) for convertingN6-acetyl-1,6-diaminohexane to N6-acetyl-6-aminohexanal was assayedusing a buffer composed of a final concentration of 50 mM HEPES buffer(pH=7.5), 10 mM N6-acetyl-1,6-diaminohexane, 10 mM pyruvate and 100 μMpyridoxyl 5′ phosphate. Each enzyme activity assay reaction wasinitiated by adding a cell free extract of the ω-transaminase or theempty vector control to the assay buffer containing theN6-acetyl-1,6-diaminohexane then incubated at 25° C. for 4 h, withshaking at 250 rpm. The formation of L-alanine was quantified viaRP-HPLC.

Each enzyme only control without N6-acetyl-1,6-diaminohexanedemonstrated low base line conversion of pyruvate to L-alanine See FIG.16.

The gene product of SEQ ID NO 7-12 accepted N6-acetyl-1,6-diaminohexaneas substrate as confirmed against the empty vector control (see FIG. 20)and synthesized N6-acetyl-6-aminohexanal as reaction product.

Given the reversibility of the ω-transaminase activity (see example 5),the gene products of SEQ ID NOs: 7-12 accept N6-acetyl-6-aminohexanal assubstrate forming N6-acetyl-1,6-diaminohexane.

Example 12 Enzyme Activity of Carboxylate Reductase Using AdipateSemialdehyde as Substrate and Forming Hexanedial

The N-terminal His-tagged carboxylate reductase of SEQ ID NO 6 (seeExample 7 and FIG. 9) was assayed using adipate semialdehyde assubstrate. The enzyme activity assay was performed in triplicate in abuffer composed of a final concentration of 50 mM HEPES buffer (pH=7.5),2 mM adipate semialdehyde, 10 mM MgCl₂, 1 mM ATP and 1 mM NADPH. Theenzyme activity assay reaction was initiated by adding purifiedcarboxylate reductase and phosphopantetheine transferase or the emptyvector control to the assay buffer containing the adipate semialdehydeand then incubated at room temperature for 20 min. The consumption ofNADPH was monitored by absorbance at 340 nm. The enzyme only controlwithout adipate semialdehyde demonstrated low base line consumption ofNADPH. See FIG. 11.

The gene product of SEQ ID NO: 6, enhanced by the gene product of sfp,accepted adipate semialdehyde as substrate as confirmed against theempty vector control (see FIG. 15) and synthesized hexanedial.

Example 13 Enzyme Activity of CYP153 Monooxygenase Using Hexanoate asSubstrate in Forming 6-Hydroxyhexanoate

A sequence encoding a HIS tag was added to the Polaromonas sp. JS666,Mycobacterium sp. HXN-1500 and Mycobacterium austroafricanum genesrespectively encoding (1) the monooxygenases (SEQ ID NOs: 13-15), (2)the associated ferredoxin reductase partner (SEQ ID NOs: 16-17) and thespecie's ferredoxin (SEQ ID NOs: 18-19). For the Mycobacteriumaustroafricanum monooxygenase, Mycobacterium sp. HXN-1500 oxidoreductaseand ferredoxin partners were used. The three modified protein partnerswere cloned into a pgBlue expression vector under a hybrid pTacpromoter. Each expression vector was transformed into a BL21[DE3] E.coli host. Each resulting recombinant E. coli strain were cultivated at37° C. in a 500 mL shake flask culture containing 50 mL LB media andantibiotic selection pressure. Each culture was induced for 24 h at 28°C. using 1 mM IPTG.

The pellet from each induced shake flask culture was harvested viacentrifugation. Each pellet was resuspended and the cells made permeableusing Y-per™ solution (ThermoScientific, Rockford, Ill.) at roomtemperature for 20 min. The permeabilized cells were held at 0° C. inthe Y-per™ solution.

Enzyme activity assays were performed in a buffer composed of a finalconcentration of 25 mM potassium phosphate buffer (pH=7.8), 1.7 mMMgSO₄, 2.5 mM NADPH and 30 mM hexanoate. Each enzyme activity assayreaction was initiated by adding a fixed mass of wet cell weight ofpermeabilized cells suspended in the Y-per™ solution to the assay buffercontaining the heptanoate and then incubated at 28° C. for 24 h, withshaking at 1400 rpm in a heating block shaker. The formation of7-hydroxyheptanoate was quantified via LC-MS.

The monooxygenase gene products of SEQ ID NO 13-15 along with reductaseand ferredoxin partners, accepted hexanoate as substrate as confirmedagainst the empty vector control (see FIG. 22) and synthesized6-hydroxyhexanoate as reaction product.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method for biosynthesizing one or more productsselected from the group consisting of adipic acid, 6-aminohexanoic acid,6-hydroxyhexanoic acid, hexamethylenediamine, caprolactam, and1,6-hexanediol, said method comprising enzymatically synthesizinghexanoyl-CoA and enzymatically forming, in one or more steps, twoterminal functional groups selected from the group consisting ofcarboxyl, amine, and hydroxyl groups in hexanoyl-CoA to produce the oneor more products.
 2. The method of claim 1, wherein said hexanoyl-CoA isproduced from acetyl-CoA in two cycles of CoA-dependent carbon chainelongation using (i) either a β-ketothiolase or an acetyl-CoAcarboxylase and an acetoacetyl-CoA synthase, (ii) a 3-hydroxyacyl-CoAdehydrogenase or a 3-oxoacyl-CoA reductase, (iii) an enoyl-CoAhydratase, (iv) and a trans-2-enoyl-CoA reductase.
 3. The method ofclaim 1, wherein said two terminal functional groups are the same orsaid two terminal functional groups are different.
 4. The method ofclaim 3, wherein said product comprises a terminal amine group and aterminal carboxyl group or a terminal hydroxyl group and a terminalcarboxyl group.
 5. The method of claim 3, wherein said two terminalfunctional groups are amine groups or said two terminal functionalgroups are hydroxyl groups.
 6. The method of claim 5, wherein aω-transaminase or a deacetylase enzymatically forms the two aminegroups.
 7. The method of claim 5, wherein a monooxygenase, anoxidoreductase and ferredoxin or an alcohol dehydrogenase enzymaticallyforms the two hydroxyl groups.
 8. The method of claim 7, wherein saidmonooxygenase has at least 70% sequence identity to any one of the aminoacid sequences set forth in SEQ ID NO. 13-15.
 9. The method of claim 1,wherein a thioesterase, an aldehyde dehydrogenase, a 7-oxoheptanoatedehydrogenase, or a 6-oxohexanoate dehydrogenase, enzymatically forms aterminal carboxyl group.
 10. The method of claim 9, wherein saidthioesterase has at least 70% sequence identity to the amino acidsequence set forth in SEQ ID NO:
 1. 11. The method of claim 4, wherein aω-transaminase enzymatically forms the amine group.
 12. The method ofclaim 11, wherein said ω-transaminase has at least 70% sequence identityto any one of the amino acid sequences set forth in SEQ ID NO. 7-12. 13.The method of claim 1, wherein a carboxylate reductase and aphosphopantetheinyl transferase form a terminal aldehyde group as anintermediate in forming the product.
 14. The method of claim 13, whereinsaid carboxylate reductase has at least 70% sequence identity to any oneof the amino acid sequences set forth in SEQ ID NO. 2-6.
 15. The methodof claim 1, wherein said method is performed in a recombinant host. 16.The method of claim 15, wherein the principal carbon source fed to thefermentation derives from biological or non-biological feedstocks. 17.The method of claim 16, wherein the biological feedstock is, or derivesfrom, monosaccharides, disaccharides, lignocellulose, hemicellulose,cellulose, lignin, levulinic acid, formic acid, triglycerides, glycerol,fatty acids, agricultural waste, condensed distillers' solubles, ormunicipal waste; or wherein the non-biological feedstock is, or derivesfrom, natural gas, syngas, CO₂/H₂, methanol, ethanol, benzoate,non-volatile residue (NVR) caustic wash waste stream from cyclohexaneoxidation processes, or terephthalic acid/isophthalic acid mixture wastestreams.
 18. The method of claim 15, wherein the host is a prokaryote.19. The method of claim 18, wherein said prokaryote is from the genusEscherichia; from the genus Clostridia; from the genus Corynebacteria;from the genus Cupriavidus; from the genus Pseudomonas; from the genusDelftia; from the genus Bacillus; from the genus Lactobacillus; from thegenus Lactococcus or from the genus Rhodococcus.
 20. The method of claim15, wherein the host is a eukaryote.
 21. The method of claim 20, whereinsaid eukaryote is from the genus Aspergillus; from the genusSaccharomyces; from the genus Pichia; from the genus Yarrowia; from thegenus Issatchenkia; from the genus Debaryomyces; from the genus Arxula;or from the genus Kluyveromyces.
 22. The method of claim 15, whereinsaid host comprises one or more of the following attenuated enzymes:polyhydroxyalkanoate synthase, an acetyl-CoA thioesterase, aphosphotransacetylase forming acetate, an acetate kinase, a lactatedehydrogenase, a menaquinol-fumarate oxidoreductase, a 2-oxoaciddecarboxylase producing isobutanol, an alcohol dehydrogenase formingethanol, a triose phosphate isomerase, a pyruvate decarboxylase, aglucose-6-phosphate isomerase, NADH-consuming transhydrogenase, anNADH-specific glutamate dehydrogenase, a NADH/NADPH-utilizing glutamatedehydrogenase, a pimeloyl-CoA dehydrogenase; an acyl-CoA dehydrogenaseaccepting C6 building blocks and central precursors as substrates; abutaryl-CoA dehydrogenase; or an adipyl-CoA synthetase.
 23. The methodof claim 15, wherein said host overexpresses one or more genes encoding:an acetyl-CoA synthetase, a 6-phosphogluconate dehydrogenase; atransketolase; a puridine nucleotide transhydrogenase; aglyceraldehyde-3P-dehydrogenase; a malic enzyme; a glucose-6-phosphatedehydrogenase; a glucose dehydrogenase; a fructose 1,6 diphosphatase; aL-alanine dehydrogenase; a L-glutamate dehydrogenase; a formatedehydrogenase; a L-glutamine synthetase; a diamine transporter; adicarboxylate transporter; and/or a multidrug transporter.
 24. Themethod of claim 4, wherein a ω-transaminase enzymatically forms theamine group.
 25. The method of claim 19, wherein said prokaryote isselected from the group consisting of Escherichia coli, Clostridiumljungdahlii, Clostridium autoethanogenum, Clostridium kluyveri,Corynebacterium glutamicum, Cupriavidus necator, Cupriavidusmetallidurans, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonasoleavorans, Delftia acidovorans, Bacillus subtillis, Lactobacillusdelbrueckii, Lactococcus lactis, and Rhodococcus equi.
 26. The method ofclaim 21, wherein said eukaryote is selected from the group consistingof Aspergillus niger, Saccharomyces cerevisiae, Pichia pastoris,Yarrowia lipolytica, Issathenkia orientalis, Debaryomyces hansenii,Arxula adenoinivorans, and Kluyveromyces lactis.